Nerium oleander oligosaccharide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicine and health care, and discloses oleander oligosaccharide and a preparation method and application thereof, the molecular weight of the oleander oligosaccharide is 2900 Da, and the oleander oligosaccharide is composed of glucose, mannose, galactose and arabinose; the preparation method comprises the following steps: degreasing oleander roots with ethanol, respectively carrying out water extraction and gradient concentration alcohol extraction, and then sequentially carrying out ceramic membrane-ultrafiltration-nanofiltration enrichment, ion exchange column chromatography and molecular sieve gel column purification on the alcohol extract to obtain the oleander extract. The oligosaccharide can be applied to preparation of immunological enhancement drugs, corresponding tumor immune drugs or health care products. The preparation method is efficient and safe, and the prepared product is good in quality and can be applied to preparation of immunological enhancement drugs, corresponding tumor immune drugs or health care products.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical and health care technology, and specifically relates to an oleander oligosaccharide and its preparation method, as well as its application in the preparation of immune-enhancing drugs or health products. Background Technology

[0002] Oleander ( Nerium oleander L. is a traditional medicinal plant. It is cold in nature, bitter in taste, and enters the heart meridian. It has the effects of strengthening the heart and promoting diuresis, relieving phlegm and asthma, relieving pain, and removing blood stasis. Modern research mainly focuses on the leaves, flowers, and bark, which are rich in various bioactive components, such as cardiac glycosides, phenols, and polysaccharides, and have multiple activities such as anti-tumor, anti-inflammatory, antioxidant, and neuroprotective effects.

[0003] Carbohydrates are one of the three basic building blocks of living organisms, possessing a wide range of biological activities and playing a vital role in many life processes. In recent years, my country has made significant progress in the research and development of polysaccharides from traditional Chinese medicine, such as Ganoderma lucidum polysaccharides, Astragalus membranaceus polysaccharides, and ginseng polysaccharides. However, the large molecular weight and low oral bioavailability of traditional polysaccharide oral medications severely limit their clinical application and the development of health products. Oligosaccharides, due to their low molecular weight, high bioavailability, and ease of quality control, are gradually becoming a research hotspot and have broad application prospects.

[0004] Currently, there is relatively little research on the sugars in oleander. A relevant study includes a Chinese patent (application number 200910111907.4) describing a method for preparing an oleander polysaccharide extract and its inhibitory effect on mouse sarcoma S180. However, this mainly focuses on the extraction process and activity study of the crude polysaccharide; the fine chemical structure of the active polysaccharide remains unclear, limiting further development. There are also reports of purifying three homogeneous polysaccharide components and evaluating their neuroprotective effects, but their chemical structures are not clearly defined (Chinese patent application number 99125746.4). Furthermore, other researchers have reported two structurally distinct oligogalactanoids purified from oleander flowers and their anti-tumor angiogenesis effects, with molecular weights of 1800 Da and 828 Da, respectively (Chinese patent application number 200810039658.8). In summary, there are currently no reports on the immune-enhancing effects of oleander oligosaccharides. Furthermore, existing patents mainly involve sugars obtained from the flowers and leaves, which have a single structural type and unclear mechanism. The purification process for oligosaccharides is cumbersome, and organic reagents used for deproteinization are prone to remain, which seriously restricts the subsequent development of oleander oligosaccharides.

[0005] To address the above deficiencies, this invention proposes a method for preparing high-purity oligosaccharides using a combination of water extraction, alcohol precipitation, and multi-stage membrane separation, which effectively reduces reaction steps and purification time. Simultaneously, the chemical structure of the active oligosaccharide is clarified, and its immune-enhancing mechanism is elucidated, laying the foundation for subsequent quality control and development in the pharmaceutical and health product fields. Summary of the Invention

[0006] The first technical problem to be solved by the present invention is to provide an oleander oligosaccharide with a molecular weight of 2900 Da, which is composed of glucose, mannose, galactose and arabinose.

[0007] The second technical problem to be solved by the present invention is to provide a method for preparing oleander oligosaccharides, which uses a combination of water extraction, alcohol precipitation and multi-stage membrane separation. The method is simple, can effectively reduce separation steps and purification time, and has good purification effect and high product purity.

[0008] The third technical problem to be solved by the present invention is to provide the application of oleander oligosaccharides in the preparation of immune-enhancing drugs, corresponding tumor immunotherapy drugs or health products.

[0009] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: An oleander oligosaccharide with a molecular weight of 2900 Da has a main chain composed of →5)-α-L-Araf-(1→,→3)-α-L-Araf-(1→,→6)-α-D-Glcp-(1→,→4)-α-D-Manp-(1→,→4)-α-D-Glcp-(1→,→4,6)-α-D-Galp-(1→ and α-L-Araf-(1→), and side chains composed of β -D-Glc p -(1→Composition, specific chemical structure is as follows:

[0010] in: Glc stands for glucose, Man for mannose, Gal for galactose, and Ara for arabinose.

[0011] This oleander oligosaccharide is a homogeneous oligosaccharide with well-preserved components, a well-defined structure, and controllable quality. It can enhance the phagocytic capacity of RAW264.7 macrophages, promote macrophage M1 polarization, and enhance the production of nitric oxide (NO) and IL-1. β Tumor necrosis factor (TNF-α) α It can release molecules such as α and β, as well as inflammatory factors, and at the same time increase the release of NO in zebrafish embryos, promote macrophage recruitment, and enhance immunity.

[0012] A method for preparing oleander oligosaccharides includes the following steps: S1. Polysaccharide extraction: Cut dried oleander roots into small sections, defatted with 95% ethanol, air-dried, added water, heated to 85 degrees Celsius and extracted for 3 hours, filtered, and the residue was extracted again with an appropriate amount of water. This process was repeated 3-4 times. The filtrates were combined, heated and concentrated to 1 / 10 volume, centrifuged, and 95% ethanol was added to the supernatant until the final ethanol concentration was 70%. The mixture was allowed to stand for 12 hours, and the precipitated polysaccharide was collected and named JZTP70. The supernatant was further concentrated, and anhydrous ethanol was added until the final ethanol concentration was 90%. The mixture was allowed to stand for 12 hours, centrifuged, and the precipitated polysaccharide was collected and named JZTP90. S2. One-step purification (rapid enrichment of oligosaccharides and removal of proteins): Dissolve the polysaccharide JZTP90 obtained in step S1 in water, remove the protein and enrich the oligosaccharides using a multi-stage membrane separation system, maintain the temperature of the feed solution at 40-55℃ and the pressure at 0.1-0.6MPa, retain the enriched oligosaccharides, and freeze-dry to obtain oligosaccharide JZTP-S. S3. Secondary purification: The oligosaccharide JZTP-S obtained in step S2 was subjected to ion exchange column chromatography, with gradient elution using water and 0.05 M, 0.10 M, 0.15 M, 0.20 M, 0.40 M, 0.8 M, and 2.0 M NaCl until no sugar fraction eluted. The elution curve was tracked using the phenol-sulfuric acid method (polysaccharide detection method) combined with HPGPC (high performance gel permeation chromatography), and the sugar fractions were collected according to the elution curves. The sugar fraction in the aqueous portion was concentrated and freeze-dried to obtain the oleander oligosaccharide in the aqueous portion, named JZTP-W. Then, take an appropriate amount of JZTP-W, dissolve it in water, centrifuge at 8000 rpm, take the supernatant for Sephadex S-100 molecular sieve gel column chromatography, elute with water, and simultaneously use the phenol-sulfuric acid method combined with HPGPC to detect the elution curve. According to the elution curve, collect the oligosaccharide fraction with good homogeneity, concentrate and freeze dry to obtain oleander oligosaccharide, named oligosaccharide JZTP-01-1.

[0013] Preferably, in step S1, the weight ratio of dried oleander root to ethanol is 1:6, and the root is soaked for 12 h for defatting. After defatting, water with a weight of 10 times that of dried oleander root is added for extraction, and the amount of water added to the residue is the same as before. Both centrifugations are performed at 10,000 rpm for 30 min.

[0014] Preferably, in step S2, the polysaccharide JZTP90 is dissolved by adding water until it is completely dissolved; the multi-stage membrane separation system is a three-stage membrane separation system, with the first stage using a ceramic membrane (pore size 0.1–0.5 mm). μThe solution is clarified (removing insoluble matter and micelle particles) and some proteins are removed using affinity separation. The effluent then enters the second stage of separation. The second stage uses a 5000 Da ultrafiltration membrane to retain large polysaccharide and protein molecules. The effluent then enters the third stage of nanofiltration. The third stage uses a 500 Da nanofiltration membrane. The effluent consists of small molecules and salts, while the retained portion is enriched oligosaccharides. Each stage of filtration involves 3-6 washes. Finally, the obtained oligosaccharide fraction is concentrated under reduced pressure (rotary evaporator) and freeze-dried to obtain JZTP-S. During the separation of materials in the multi-stage membrane separation system, the feed temperature is maintained at 50°C and the pressure at 0.5 MPa.

[0015] Preferably, in step S3, the material is dissolved in 30 times its weight of water, centrifuged at 8000 rpm for 10 min, concentrated under reduced pressure at a temperature of 40–70°C (pressure of 800 Pa), and then freeze-dried (-65°C, vacuum degree 10–20 Pa).

[0016] Preferably, the oleander oligosaccharide is detected using the following methods: 1. Take an oleander oligosaccharide sample, completely acid-hydrolyze it, and detect the hydrolysis product PMP (1-phenyl-3-methyl-5-pyrazolone) by liquid chromatography after derivatization; 2. Take an oleander oligosaccharide sample, dry it, compress it into a tablet, and detect it by infrared spectroscopy; 3. Take an oleander oligosaccharide sample, methylate it, hydrolyze it, reduce it, acetylate it, and perform GC-MS (…). Gas Chromatography-Mass Spectrometry 4. Dissolve oleander oligosaccharide samples in D2O (heavy water) and perform nuclear magnetic resonance analysis.

[0017] Applications of oleander oligosaccharides: Oleander oligosaccharides are used in the preparation of immune-enhancing drugs, corresponding tumor immunotherapy drugs, or health products.

[0018] Due to the adoption of the above technical solution, the beneficial effects of the present invention are: 1. This invention first extracts polysaccharides from the rhizomes of oleander using water, then fractionates them with high-concentration ethanol to obtain oligosaccharides with higher polarity. A multi-stage ethanol precipitation-multi-stage membrane separation method is then used to achieve rapid impurity removal and enrichment of oleander oligosaccharides. The method is effective and more environmentally friendly and efficient than traditional methods, avoiding the biosafety issues of organic solvents. It also has high separation throughput, wider applicability, and is suitable for large-scale production. 2. This invention uses column chromatography to perform secondary separation and purification of oleander oligosaccharides, with significant results, and prepares a homogeneous oleander oligosaccharide with a different structure from existing ones. 3. The oleander oligosaccharide obtained by this invention has well-preserved components, a clear structure, and controllable quality. This oligosaccharide can enhance the phagocytic ability of RAW264.7 macrophages, promote macrophage polarization towards the M1 phenotype, and promote the release of inflammatory factors such as nitric oxide (NO), IL-1β, and tumor necrosis factor (TNF)-α. At the same time, it can increase the release of NO in zebrafish embryos, promote macrophage recruitment, and enhance immunity. It can be applied in the fields of immune-enhancing drugs, corresponding tumor immunotherapy drugs, and health products. Attached Figure Description

[0019] Figure 1 HPGPC spectra of homogenous oligosaccharide JZTP-01-1 and molecular weight analysis. Figure 2 High-performance liquid chromatogram of oligosaccharide JZTP-01-1 monosaccharide composition; Figure 3 Infrared spectrum of oligosaccharide JZTP-01-1; Figure 4 For oligosaccharide JZTP-01-1 1 H-map; where A is the full map and B is a magnified view of the full map; Figure 5 For oligosaccharide JZTP-01-1 13 C10 NMR spectra; where C is the full spectrum and D is a magnified view of a portion. Figure 6 The image shows the HSQC spectrum of oligosaccharide JZTP-01-1; where A is the full spectrum and B is a magnified view. Figure 7 The image shows the HMBC spectrum of oligosaccharide JZTP-01-1; where A is the full spectrum and B is a magnified view of a portion of the spectrum. Figure 8 It represents the primary structure of oligosaccharide JZTP-01-1; Figure 9 The effects of oligosaccharide JZTP-01-1 on the cytotoxicity, phagocytic capacity, inflammatory factors, and chemokine levels of RAW264.7 cells; Figure 10 The effects of oligosaccharide JZTP-01-1 on the secretion levels of inflammatory factors and the transcription levels of chemokines in RAW264.7 cells; Figure 11 The killing effect of oligosaccharide JZTP-01-1 on tumor cells under co-culture conditions; Figure 12 The promoting effect of oligosaccharide JZTP-01-1 on the recruitment of immunosuppressive zebrafish macrophages and neutrophils; Figure 13The study investigated the NO-promoting (AB) and anti-angiogenic (CD) effects of oligosaccharide JZTP-01-1 on immunosuppressed zebrafish. Detailed Implementation

[0020] The present invention will be further illustrated below with reference to the embodiments.

[0021] Example 1: Extraction of Oleander Oligosaccharides S1. Polysaccharide Extraction: Oleander roots were cut into small sections and defatted with 95% ethanol (6 times the weight of the dried roots) for 12 hours. The root sections were then removed, dried, and extracted with water (10 times the weight of the dried roots) at 85°C for 3 hours. The mixture was filtered, and the residue was extracted again with water, repeated four times. All filtrates were combined and concentrated under vacuum at 70°C to 1 / 10 of the original volume. The mixture was centrifuged, and the supernatant was added with 95% ethanol to a final concentration of 70%. The mixture was allowed to stand for 12 hours, and the precipitated polysaccharide was collected and named JZTP70 (to be processed separately). The supernatant was further concentrated to 1 / 10 of its original volume, and anhydrous ethanol was added to a final concentration of 90%. The mixture was allowed to stand for 12 hours, and centrifuged at 10,000 rpm for 30 minutes. The precipitated polysaccharide was collected and named JZTP90.

[0022] S2. Primary purification: A multi-stage membrane separation system (ceramic membrane-ultrafiltration-nanofiltration) is used to further remove proteins and enrich oligosaccharides. The feed temperature is maintained at 50℃ and the pressure at 0.5 MPa throughout the process.

[0023] Dissolve the JZTP90 obtained in step S1 in 30 times its volume of water, then add it to the material tank and pump it in through a material pump with an orifice diameter of 0.4 mm. μ The first stage uses a ceramic membrane of 0.5 MPa to remove insoluble matter and micelle particles. The membrane is washed three times with water, and the effluent enters the secondary ultrafiltration material tank. The second stage uses a 5000 Da ultrafiltration membrane to remove large polysaccharides and proteins. It is washed three times, and the effluent enters the tertiary separation material tank. The third stage uses a 500 Da nanofiltration membrane to retain the oligosaccharides to be enriched. The effluent consists of small molecules and salts. It is washed three times. The enriched oligosaccharides were concentrated using a rotary evaporator (65°C) and freeze-dried (-65°C, vacuum 15 Pa) to obtain oligosaccharides named JZTP-S oligosaccharides.

[0024] S3. Secondary purification: 1) Ion exchange column chromatography: Take 5 g of JZTP-S oligosaccharide obtained in step S2, dissolve it in 50 mL of deionized water, load it onto a DEAE-FF column, and perform gradient elution with water and 0.05 M, 0.10 M, 0.15 M, 0.20 M, 0.40 M, 0.8 M, and 2.0 M NaCl in sequence. The elution peak of the water fraction is the target sugar fraction (the elution curve is tracked using the phenol-sulfuric acid method during the elution process, and the sugar fraction is collected according to the elution curve). The eluent of the target sugar fraction is concentrated under reduced pressure (rotary evaporator, 70℃, 800 Pa) and freeze-dried. The product obtained is named oligosaccharide JZTP-01. 2) Molecular sieve gel chromatography: The above-mentioned lyophilized oligosaccharide JZTP-01 was dissolved in 30 times the amount of water, centrifuged at 8000 rpm for 15 minutes, and the supernatant was collected and loaded onto a Sephadex G-75 column. The column was eluted with water, and the elution curve was tracked using the phenol-sulfuric acid method and detected by HPGPC. The sugar fraction at the main peak was collected, concentrated under reduced pressure (70℃, 800 Pa), and lyophilized to obtain the product oleander oligosaccharide, which was named oligosaccharide JZTP-01-1.

[0025] Example 2: Structural Analysis of Oligosaccharide JZTP-01-1 Experimental material: Oligosaccharide JZTP-01-1; Specific experimental method: 1. Molecular weight and homogeneity analysis The relative molecular weight of oligosaccharide JZTP-01-1 was determined by high performance gel permeation chromatography (HPGPC). The basic principle is that when a homogeneous polysaccharide passes through gel permeation chromatography, it forms symmetrical chromatographic peaks. The elution time is related to the molecular weight, and the calculation is performed based on the calibration curve obtained from the known molecular weight.

[0026] Chromatographic conditions: Separation was performed using a TSK GMPWXL gel column; the mobile phase flow rate was 0.8 mL / min; and the injection volume was 20 μL. μ L, ultrapure water as the mobile phase, column temperature 25℃, detector is a differential detector (RID).

[0027] Accurately weigh 2.0 mg each of oligosaccharide JZTP-01-1 and Dextrans series standards, and prepare a 2.0 mg / mL solution with ultrapure water. Before injection, use 0.45... μ After filtration through a microporous membrane, the sample was analyzed, and the retention time was recorded. A standard curve was plotted with the logarithm of the standard molecular weight (Lg) on ​​the ordinate and the retention time on the abscissa. The corresponding linear regression equation was obtained, and the relative molecular weight of oligosaccharide JZTP-01-1 was calculated. The obtained HPGPC spectrum is shown below. Figure 1 .

[0028] 2. Monosaccharide composition analysis Sample processing: Accurately weigh 4.0 mg of each oleander oligosaccharide JZTP-01-1 sample into a stoppered test tube, add 2.0 mL of 2 M trifluoroacetic acid (TFA), and place in an oil bath at 120℃ for 6 h for hydrolysis. Cool to room temperature, repeatedly add methanol and evaporate to dryness to remove TFA, dissolve in deionized water to 1 mL, centrifuge at 4000 rpm for 10 min, and take 100 μL of each sample. μ L sample solution, add 100 μ Add 100 L of 0.3 M NaOH solution. μ Mix 0.5 M PMP (1-phenyl-3-methyl-5-pyrazolone) methanol solution, react in a 70 °C water bath for 30 min, cool to room temperature, and add 105 L μ Neutralize with 0.3 M HCl solution, add deionized water to 1 mL, then add an equal volume of chloroform solution, shake vigorously, centrifuge at 4000 rpm for 5 min, remove the chloroform phase, repeat the extraction process twice, and use the aqueous phase for 0.22 L… μ The sample is filtered through an m-filter membrane and then injected for HPLC analysis.

[0029] Chromatographic conditions: Column: Kromasil 100-5-C18, 4.6 × 250 mm, 5 μ m; Mobile phase: 0.1 M phosphate (pH=6.9) buffer-acetonitrile (v / v 83:17); Detection wavelength: 250 nm; Flow rate: 0.8 mL / min; Injection volume: 20 μ L. The obtained high-performance liquid chromatography chromatogram is shown in the figure. Figure 2 .

[0030] 3. Infrared spectroscopy detection 2.0 mg of dried oligosaccharide JZTP-01-1 was ground and mixed with potassium bromide, then compressed into tablets and heated using a Perkin EImerFT / IR-100 at 4000–400 cm⁻¹. -1 A scan was performed within the specified range. The resulting atlas can be found in [the image / data]. Figure 3 .

[0031] 4. Methylation / GC-MS Analysis Weigh 8.0 mg of dried oligosaccharide JZTP-01-1 into a reaction flask, add 8 mL of anhydrous DMSO (dimethyl sulfoxide), then add 800 mg of dried sodium hydroxide, sonicate at 160 Hz for 30 min, and add 3.0 mL of iodomethane in an ice-water bath in the dark (add in three portions, 1.0 mL each time, sonicating at 160 Hz for 30 min after each addition). After the reaction is complete, add 2 mL of distilled water to decompose the residual iodomethane, and add 1 mL of chloroform for extraction. After centrifugation at 4000 rpm for 10 min, collect the chloroform layer to obtain methylated oleander oligosaccharide.

[0032] Methylated oleander oligosaccharides were placed in a stoppered test tube, and hydrolyzed in a 120°C oil bath with 2 mol / L TFA (trifluoroacetic acid) for 6 h. The mixture was then evaporated to dryness under reduced pressure using a rotary evaporator at 60°C and 800 Pa. This process was repeated 2–4 times with the addition of 4 mL of methanol, followed by rotary evaporation until the pH was neutral (excess TFA was removed by rotary evaporation; the product without TFA is neutral). The hydrolysate was then reduced by reacting with 20 mg of NaBH4 at 40°C for 30 min. Finally, 100 mg of NaBH4 was used to reduce the hydrolysis product. μ The reaction was terminated with glacial acetic acid of L. The sample was evaporated to dryness under reduced pressure using a rotary evaporator. Then, 2 mL each of acetic anhydride and pyridine were added sequentially for acetylation. The reaction was maintained at 95 °C with magnetic stirring for 2 h. Methanol was added repeatedly three times, and the sample was evaporated to dryness using a rotary evaporator. The sample was dissolved in 1 mL of chloroform and washed three times with an equal volume of distilled water to remove the aqueous layer. Finally, the chloroform layer was dried with anhydrous sodium sulfate, and the sodium sulfate solid was removed by filtration. The filtrate was concentrated to dryness under reduced pressure using a rotary evaporator and analyzed by GC-MS (Thermo TSQ9610).

[0033] 5. Nuclear magnetic resonance analysis After repeatedly lyophilizing the oligosaccharide sample JZTP-01-1, 60 mg was dissolved in 0.6 mL of D2O, placed in an NMR tube, and recorded using a Bruker AV-600 NMR spectrometer at 600 MHz. 1 H NMR, 13 C10 NMR, HSQC, and HMBC spectra. See [link to spectrum]. Figures 4 to 7 .

[0034] Experimental results: 1. Structural analysis of oligosaccharide JZTP-01-1 (1) Analysis of molecular weight, homogeneity and monosaccharide composition like Figure 1 As shown in the HPGPC spectrum, oligosaccharide JZTP-01-1 has a single symmetrical peak and a molecular weight of 2900 Da, indicating that it is a homogeneous oligosaccharide.

[0035] like Figure 2 As shown in the HPLC chromatogram, the oligosaccharide JZTP-01-1 is composed of mannose, glucose, galactose, and arabinose, with a molar ratio of 35.3:41.9:5.3:17.5. (Chromatographic peak order: 1: mannose, 2: rhamnose, 3: glucuronic acid, 4: galacturonic acid, 5: glucose, 6: galactose, 7: xylose, 8: arabinose, 9: fucose) (2) Infrared spectroscopy analysis like Figure 3 As shown in the infrared spectrum, oligosaccharide JZTP-01-1 contains the characteristic infrared absorption peaks of sugars.

[0036] (3) Methylation / GC-MS analysis Methylation analysis of oligosaccharide JZTP-01-1 was performed. After hydrolysis and reductive acetylation, GC-MS detection was conducted using a Thermo TSQ9610 mass spectrometer. The results showed that oligosaccharide JZTP-01-1 contained (1→5)-α-L-Araf-(1→,→3)-α-L-Araf-(1→,→6)-α-D-Glcp-(1→,→4)-α-D-Manp-(1→,→4)-α-D-Glcp-(1→,→4,6)-α-D-Galp-(1→ and α-L-Araf ... β -D-Glc p -(1→ isoglycolic residues, the molar ratio of each sugar residue from front to back is 6.13:7.89:3.71:29.88:30.31:7.52:9.91:4.63.

[0037] (4) Nuclear magnetic resonance analysis This experiment passed 1 H NMR, 13 The chemical shifts of carbon and hydrogen atoms of the sugar residues in oligosaccharide JZTP-01-1 were assigned by C NMR and HSQC, and then the linkage sequence was confirmed by HMBC. Figures 4-7 These are oligosaccharides JZTP-01-1 1 HNMR and 13 C NMR, HSQC, and HMBC spectra.

[0038] In summary, oligosaccharide JZTP-01-1 is a glucomannan oligosaccharide mainly composed of glucose and mannose. Methylation analysis indicates that it contains →5)-α-L-Araf-(1→,→3)-α-L-Araf-(1→,→6)-α-D-Glcp-(1,→4)-α-D-Manp-(1→,→4)-α-D-Glcp-(1→,→4,6)-α-D-Galp-(1→ and α-L-Araf-(1→ andβ -D-Glc p -(1→Isoglycolic residues, the connection order between different sugar residues was determined by two-dimensional NMR HMBC spectrum analysis. Based on the above analysis, the primary structure of oligosaccharide JZTP-01-1 is as follows: Figure 8 As shown.

[0039] Example 3: Immunomodulatory effect of oligosaccharide JZTP-01-1 on macrophages Experimental material: Oligosaccharide JZTP-01-1 ； Experimental subject: RAW264.7 cells (provided by the Shanghai Cell Bank, Chinese Academy of Sciences); Specific experimental methods: 1. Experimental grouping and drug administration: This invention utilizes a RAW264.7 macrophage model, and includes a blank control group, a positive drug group, and an oligosaccharide JZTP-01-1 administration group, wherein the concentration of oligosaccharide JZTP-01-1 is 50%. μ g / mL, 200 μ g / mL and 400 μ g / mL, the positive control agent is lipopolysaccharide (LPS), and the concentration is 1 g / mL. μ g / mL, the blank control group was sterile water, and the added volume was 200 g / mL. μ L.

[0040] Take mouse macrophages RAW264.7 cultured in a culture dish, discard the culture medium, and add PBS (phosphate-coated PBS). buffer Wash cells with saline solution, add 1 mL of trypsin to digest cells for 1 min, prepare a homogeneous single-cell suspension and seed in a 96-well plate, adjusting the cell concentration to 5 × 10⁶ cells / well. 4 Cells / mL. After incubation at 37℃ for 24h, add positive control drug lipopolysaccharide, oligosaccharide JZTP-01-1 and sterile water at the above concentration. After administration, place in a CO2 incubator and incubate at 37℃ for 24h.

[0041] 2. Cytotoxicity assay After incubation for 48 h in a 96-well plate following drug administration, 10 mg of the drug was added to each well. μ L of CCK-8 solution (Cell Counting Kit) -8 Cell counting reagents were used, and the cells were incubated at 37°C for 2 hours in a CO2 incubator. The absorbance (OD value) at 450 nm was then measured using a microplate reader. Cell viability was calculated for different groups according to the CCK-8 kit instructions. The experiment was repeated three times.

[0042] 3. Neutral red phagocytosis experiment RAW264.7 cells were treated with a blank control, lipopolysaccharide, and different concentrations of oligosaccharide JZTP-01-1, followed by washing three times with PBS, and then 100 mg / L of each solution was added. μ Incubate with 0.1% neutral red solution for 1 h, discard the neutral red solution, wash three times with PBS, and aspirate dry; then add 150 μL of cell lysis buffer (glacial acetic acid: ethanol = 1:1). μ L, incubated at room temperature for 1 h, and the OD value was measured at 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The experiment was repeated three times.

[0043] 4. Effects of oligosaccharide JZTP-01-1 on inflammatory factors and chemokines in RAW264.7 cells RAW264.7 cells were seeded into 24-well plates and cultured in a cell culture incubator for 4 h. Then, blank control, lipopolysaccharide, and oligosaccharide JZTP-01-1 (50 mg / L) were added respectively. μ g / mL, 200 μ g / mL and 400 μ g / mL) and 1 μ After treatment with 1000 g / mL LPS for 24 h, the supernatant was collected and TNF-α was detected according to the ELISA kit instructions. α and IL-1 β The levels of inflammatory factors were measured. After 24 h of cell treatment, cells were collected and RNA was extracted. TNF-α levels were detected by RT-PCR. α transcriptional levels of iNOS, CXCL1, CCL2, and chemokines.

[0044] 5. The promoting effect of oligosaccharide JZTP-01-1 on M1 polarization in RAW264.7 cells RAW264.7 cells were seeded in 12-well plates and cultured for 6 h. Afterward, cells were administered either a blank control group (sterile water) or lipopolysaccharide (LPS 1). μ g / mL), different concentrations of oligosaccharide JZTP-01-1 (50 g / mL), μ g / mL, 200 μ g / mL and 400 μ (g / mL), continue culturing in the cell culture incubator for 24 h, discard the supernatant, and add 500 g / mL of 4% paraformaldehyde. μ Cells were fixed for 20 min, washed with PBS, and then 500 ml of 0.1% Triton X-100 solution was added. μ Permeabilize for 5 minutes, then add 500 ml of water. μ After blocking with 5% BSA, add 100 drops. μ L-antibody (CD80, iNOS) was incubated at room temperature for 12 h, and then 100 μL of the solution was added under light-protected conditions. μL-goat anti-rabbit fluorescent secondary antibody IgG was incubated at room temperature for 1 h, mounted, and the fluorescence intensity was observed and analyzed using an Olympus VS200 slide scanner.

[0045] RAW264.7 cells were seeded in 12-well plates and cultured for 6 h. Afterward, cells were administered either a blank control group (sterile water) or lipopolysaccharide (LPS 1). μ g / mL), different concentrations of oligosaccharide JZTP-01-1 (50 g / mL), μ g / mL, 200 μ g / mL and 400 μ (g / mL), continue culturing in the cell culture incubator for 24 h, discard the supernatant and scrape off the cells with a cell scraper, centrifuge at 300 g for 5 min to collect the cells, wash twice with PBS, and add 20 g / mL under the dark. μ Fluorescently labeled antibodies (CD86-FITC, CD206-PE) diluted in L staining buffer (100:1) were incubated at room temperature for 15 min, resuspended in a shaker, and then 200 μL of the solution was added. μ L staining buffer was used to analyze the positive rate of CD86 using a CytoFLEX S analyzer to determine the effect of oligosaccharide JZTP-01-1 on promoting M1 polarization.

[0046] 6. Antitumor effects of oligosaccharide JZTP-01-1 The study used a co-culture method between RAW264.7 cells and human breast cancer cells MDA-MB-231 to investigate whether the oligosaccharide JZTP-01-1 exerts its anti-tumor effect by promoting macrophage polarization.

[0047] RAW264.7 cells were seeded in Transwell chambers (0.4 g / cm³). μ The upper layer of the chamber (with a pore size of 5 μm) was incubated with human breast cancer cells MDA-MB-231 seeded in the lower layer, avoiding direct contact but allowing cytokine exchange. After culturing in the cell incubator for 6 h, different concentrations of oligosaccharide JZTP-01-1 (50 μL) were introduced into the upper chamber. μ g / mL, 200 μ g / mL and 400 μ The cells were treated with a medium (g / mL) for 24 h, with the lower layer of cells left untreated. A blank control group was also included. After 24 h, the culture medium was discarded, the cells were washed with PBS, and 200 mg / mL of the solution was added. μ L4% paraformaldehyde fixed lower layer human breast cancer cells, then added 500 μStain with 0.1% crystal violet for 15 min, then wash off the staining solution with PBS, observe and photograph under a Leica microscope. After photographing, add 0.5 mL of 10% acetic acid to each well, shake for 15 min to fully dissolve the adsorbed crystal violet, forming a homogeneous purple solution, and measure the OD value at 570 nm using a microplate reader. Analyze and statistically analyze the results.

[0048] Experimental results: 1. Effects of oligosaccharide JZTP-01-1 on macrophage cytotoxicity (results shown in Table 1), phagocytic capacity (results shown in Table 2), inflammatory factors (results shown in Table 3), and chemokine secretion levels (results shown in Table 4).

[0049] Table 1. Comparison of cytotoxicity results of oligosaccharide JZTP-01-1 and blank control against RAW264.7 macrophages.

[0050] like Figure 9 As shown in Table 1, after treatment with different concentrations of oligosaccharide JZTP-01-1, the activity and cell viability of RAW264.7 cells were basically the same as those of the blank control group. High concentrations showed a slight proliferative effect, but no significant difference was observed. P >0.05), indicating that oligosaccharide JZTP-01-1 is at 50 µ g / mL~400 µ It is safe and non-cytotoxic within the g / mL range.

[0051] Table 2 Comparison of phagocytic capacity of oligosaccharide JZTP-01-1, blank control, and positive control on RAW264.7 macrophages.

[0052] * Significant difference compared to the blank control. P <0.05;** Significant difference compared to the blank control. P <0.01.

[0053] The test results of macrophage phagocytic capacity are as follows: Figure 9 As shown in Table B and Table 2, compared with the blank control group, the phagocytic capacity of the positive drug group was significantly increased by 1.8 times ( ). P <0.05), after administration of different doses of oligosaccharide JZTP-01-1, the phagocytic capacity was 1.2 times, 1.3 times, and 1.5 times that of the blank control group, respectively. P <0.05, P <0.05, P <0.01), with the high-dose group differing from the positive control group by only 30%.

[0054] Table 3. Experimental results of oligosaccharide JZTP-01-1, blank control, and positive control on RAW264.7 inflammatory factors.

[0055] * Significant difference compared to the blank control. P <0.05;** Significant difference compared to the blank control. P <0.01; *** Significant difference compared to the blank control. P <0.001.

[0056] Since the levels of inflammatory factors and chemokines are also standards for testing whether immune enhancement functions are present, this invention detects the TNF-α levels in RAW264.7 mouse macrophages after treatment with oligosaccharide JZTP-01-1. α IL-1 β The secretion and transcription levels. For example... Figure 9 C, Figure 9 As shown in Table 3, compared with the blank control group, TNF-α levels decreased after LPS treatment. α The secretion and transcription levels increased by 2.22-fold and 6.50-fold, respectively. P <0.001, P <0.001), low, medium and high doses of oligosaccharide JZTP-01-1 group TNF- α The secretion and transcription levels were upregulated to 1.88-fold, 2.63-fold, and 2.93-fold, respectively. P <0.01, P <0.001, P <0.001) and 2.5 times, 5.1 times, 4.8 times ( P <0.01, P <0.001, P <0.001). Among them, the high-dose oligosaccharide JZTP-01-1 group showed 91% and 64% of the LPS group, respectively. IL-1 β secretion levels such as Figure 9 As shown in Table 3, compared with the blank control group, LPS significantly increased its secretion level (2.98 times). P <0.001). IL-1 levels after treatment with different concentrations of oligosaccharide JZTP-01-1. β The secretion levels increased by 1.88 times, 2.23 times, and 2.52 times, respectively. P <0.01, P <0.001, P <0.001). All of the above results indicate that oligosaccharide JZTP01-1 can significantly promote macrophage immune activation.

[0057] Table 4. Experimental results of oligosaccharide JZTP-01-1, blank control, and positive control on M1 type chemokine in RAW264.7 cells.

[0058] * Significant difference compared to the blank control. P <0.05;** Significant difference compared to the blank control. P <0.01; *** Significant difference compared to the blank control. P <0.001.

[0059] The effect of oligosaccharide JZTP-01-1 on the transcriptional level of M-type chemokines in RAW264.7 cells is as follows: Figure 9 China F- Figure 9 As shown in Table 4, compared with the blank control group, the LPS group significantly promoted the expression of macrophage M1 chemokines iNOS, CXCL1, and CCL2. P <0.001, P <0.001, P <0.001). After treatment with different concentrations of oligosaccharide JZTP-01-1, the expression levels of the three chemokines were significantly increased ( P <0.01, P <0.001, P <0.001). Among them, 400 μ After treatment with oligosaccharide JZTP-01-1 at g / mL, the transcriptional levels of iNOS and CCL2 were comparable to those in the LPS group, while the transcriptional level of CXCL1 was significantly higher than that in the LPS group. P <0.001). The above results indicate that oligosaccharide JZTP01-1 can significantly promote the expression of macrophage M1 factor and enhance the body's immune level.

[0060] 2. Oligosaccharide JZTP-01-1 promotes macrophage M1 polarization. like Figure 10 China A- Figure 10 As shown in Figure C, compared with the blank control group, the LPS group significantly promoted the expression of CD80 and iNOS. P <0.001, P <0.001). Treatment with different concentrations of oligosaccharide JZTP-01-1 significantly upregulated the expression of the M1 phenotype protein CD80. P <0.01, P <0.001, P<0.01), which were 1.45 times, 1.79 times, and 1.57 times that of the blank control group, respectively, of which 200 μ The g / mL dose group was significantly more effective than the positive control group (1.66-fold). Similarly, iNOS protein expression was also significantly increased by 1.71-fold, 1.77-fold, and 1.87-fold, with the high-dose oligosaccharide JZTP-01-1 group being significantly superior to the positive control group (LPS). P <0.01).

[0061] At the same time, such as Figure 10 D- Figure 10 According to flow cytometry analysis, compared with the blank control group, treatment with all three doses of oligosaccharide JZTP-01-1 significantly promoted the expression ratio of the M1 phenotype protein CD86. P <0.001, P <0.001, P The value was <0.001, consistent with the results of CD80 and iNOS protein analysis, while the proportion of CD206, a marker protein of M2 macrophages, did not change significantly, indicating that oligosaccharide JZTP-01-1 can exert an immune-enhancing effect by promoting M1 macrophage polarization.

[0062] 3. Oligosaccharide JZTP-01-1 exerts its anti-tumor effect by promoting M1 polarization of macrophages. The OD values ​​of RAW264.7 cells after treatment with different concentrations of oligosaccharide JZTP-01-1 are shown in Table 5.

[0063] like Figure 11 Table 5 shows the results (blank control without JZTP-01-1, JZTP-01-1-50 is the concentration added at 50 mg / L). μ The sample group was 200 g / mL, and JZTP-01-1-200 was the spiking concentration. μ The sample group was 400 g / mL, and JZTP-01-1-400 was the spiking concentration. μ In sample groups of g / mL, RAW264.7 cells treated with different concentrations of oligosaccharide JZTP-01-1 showed significantly reduced survival rates and OD values ​​in the lower Transwell chambers of human breast cancer cells. P <0.001, P <0.001, P The value <0.001 indicates that the oligosaccharide JZTP-01-1 can exert an anti-tumor effect by promoting the polarization of macrophage M1 cells.

[0064] Table 5. The indirect effect of oligosaccharide JZTP-01-1 on tumor cell growth by regulating macrophage M1 polarization.

[0065] * Significant difference compared to the blank control. P <0.05;** Significant difference compared to the blank control. P <0.01; *** Significant difference compared to the blank control. P <0.001.

[0066] In summary, the in vitro experimental results indicate that the oligosaccharide JZTP-01-1 not only possesses direct immunomodulatory activity but also demonstrates the potential to indirectly inhibit tumors by regulating the immune system. 。

[0067] Example 4 Immunomodulatory effects of oligosaccharide JZTP-01-1 on zebrafish embryos The specific experimental method is as follows: 1. Collection and culture of zebrafish embryos The diurnal rhythm of adult zebrafish was controlled, with the day-night cycle set at 14 h:10 h. Fertilized zebrafish eggs were collected, placed in a petri dish, and sea salt medium (0.2% instant sea salt dissolved in deionized water) was added. The dish was then placed in a constant temperature incubator at 28.5℃.

[0068] 2. Experimental Design and Grouping Zebrafish embryos, 7–8 hours after fertilization, were placed in 12-well plates with 6 juvenile zebrafish per well. Control, model / positive control, and drug administration groups were established. The control group received only sea salt culture medium. The model and positive control groups were set up according to different activity tests. In the immunosuppression model, macrophage and neutrophil recruitment experiments used NVB (vinorelbine) for modeling, forming the model group; NO secretion experiments used CAP (chloramphenicol) for modeling, forming the CAP model group; and angiogenesis inhibition experiments used the vascular endothelial growth factor receptor inhibitor PTK787 (vataraniline) as the positive control, forming the positive control group. The drug administration groups received different concentrations of oligosaccharide JZTP-01-1 (50 mg / L) after modeling. μ g / mL, 200 μ g / mL, 400 μ The sample was treated at 28.5℃ for 24 h (g / mL).

[0069] 3. In vivo fluorescence imaging observation of zebrafish (1) Effect of oligosaccharide JZTP-01-1 on the recruitment of zebrafish macrophages To facilitate observation of zebrafish macrophages, E3 medium supplemented with the melanin production inhibitor PTU (N-phenylthiourea) was used throughout the experiment, with a final PTU concentration of 0.2 mM. Following the method described in the experimental design and grouping section, the drug solution was discarded 24 hours later, and the juvenile fish were washed with E3 medium containing PTU. They were then cultured for another 6 hours in the dark with 300 μL of 10 μg / mL neutral red solution. After staining, the juvenile fish were anesthetized with 0.02% MS222 (tricaine), and then placed on agarose gels. Head macrophages were observed and photographed using a stereomicroscope for statistical analysis.

[0070] (2) The recruitment effect of oligosaccharide JZTP-01-1 on zebrafish neutrophils After administering the drug 24 hours according to the method provided in the experimental design and grouping section, the drug solution was discarded. Then, the juvenile fish were anesthetized with 0.02% MS222, placed on agarose, and the red fluorescently labeled neutrophils in the tail were observed directly through a stereomicroscope. The relative fluorescence intensity in the zebrafish juveniles was observed and photographed under a laser confocal microscope.

[0071] (3) Nitric oxide (NO) release assay Following the methods described in the experimental design and grouping section, after 24 h of drug administration, the culture medium containing oligosaccharide JZTP-01-1 was removed and replaced with fresh sea salt medium periodically. Culture continued until 72 h post-fertilization of zebrafish larvae, during which the zebrafish were not fed. After treatment, the NO fluorescent probe DAF-FMDA (5 μM) was added to the culture medium and incubated in the dark for another 2 h. After incubation, the zebrafish larvae were rinsed three times with water, then anesthetized with 0.02% tricaine solution and fixed with 1 mL of 3% methylcellulose. After fixation, the relative fluorescence intensity in the zebrafish larvae was observed and photographed under a laser confocal microscope. ImageJ software was used to quantitatively analyze the relative fluorescence intensity in the zebrafish larvae to detect the effect of oligosaccharide JZTP-01-1 on NO release in zebrafish.

[0072] (4) Anti-angiogenic activity assay Transgenic Tg(fli1:EGFP) zebrafish eggs with normal fertilization at 22 hpf were selected, demeasuring and transferred to 24-well plates with 20 eggs per well. The fish were administered drugs according to the grouping and administration protocol provided in the experimental design and grouping section. After treatment at 72 hpf in an incubator (14 h light / 10 h dark cycle, 28.5℃), the juvenile fish were anesthetized with 0.02% MS222 and fixed with 1.2% low-melting-point agarose at 25℃. The fish were photographed under a fluorescence microscope, and the fluorescence intensity of the intersegmental vessels (ISVs) was quantified.

[0073] Experimental results 1. Immunoenhancing effect of oligosaccharide JZTP-01-1 on an immunosuppressed model zebrafish like Figure 12 China A- Figure 12 As shown in Figure D, compared with the control group, the addition of the immunosuppressant NVB (vinorelbine) significantly reduced the number of macrophages and neutrophils in zebrafish. P <0.001, P <0.001), indicating that the zebrafish immunosuppression model was successfully established. With increasing concentrations of oligosaccharide JZTP-01-1 (50... μ g / mL, 200 μ g / mL, 400 μ The number of macrophages in the head of zebrafish was 3.23 times, 3.38 times, and 3.68 times that of the NVB group, respectively, and the fluorescence intensity of neutrophils in the tail increased by 0.9 times, 1.5 times, and 1.56 times, respectively.

[0074] Similarly, as Figure 13 China A- Figure 13 As shown in Figure B, the fluorescence intensity of NO in the CAP group was significantly lower than that in the control group. P <0.001), indicating that the zebrafish immunosuppression model was successfully established. Different concentrations of J oligosaccharide ZTP-01-1 (50 μL) were used to treat the zebrafish. μ g / mL, 200 μ g / mL, 400 μ After treatment with g / mL, the fluorescence intensity of NO increased by 3.12 times, 3.20 times and 3.51 times, respectively.

[0075] The above results indicate that oligosaccharide JZTP-01-1 significantly promotes the recruitment of macrophages and neutrophils and the secretion of NO in zebrafish larvae, meaning that oligosaccharide JZTP-01-1 can enhance the immune capacity of zebrafish.

[0076] 2. Inhibitory effect of oligosaccharide JZTP-01-1 on angiogenesis In vitro cell experiments showed that the oligosaccharide JZTP-01-1 could indirectly inhibit tumor cell proliferation by promoting the M1 transformation of macrophages. To further evaluate its anti-tumor effect, we examined its effect on angiogenesis, and the results are as follows: Figure 13 C- Figure 13 As shown in Figure D. Compared with the control group, the fluorescence intensity of interstitial vessels was significantly reduced after treatment with different doses of oligosaccharide JZTP-01-1 ( ). P <0.001, P <0.001, P<0.001), which were 47.2%, 45.4% and 37.1% of the control group, respectively. It was only 22.2% different from the positive drug PTK787 (vatarani base) (14.9% of the positive drug), showing a significant inhibitory effect on angiogenesis, suggesting that it has potential anti-tumor effects.

[0077] In summary, the oligosaccharide JZTP-01-1 prepared in this invention can enhance the body's immunity by increasing the phagocytic capacity of RAW246.7 cells, promoting macrophage M1 polarization, and upregulating the release of related inflammatory factors. At the same time, oligosaccharide JZTP-01-1 can increase the recruitment of macrophages and neutrophils in zebrafish larvae and promote NO release.

[0078] Because of the immune-enhancing ability of oligosaccharide JZTP-01-1, the oleander oligosaccharide obtained in this invention can be used as an immune-enhancing drug or a corresponding tumor immunotherapy drug, or as a raw material for the preparation of immune-enhancing health products.

[0079] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An oleander oligosaccharide, characterized in that: Oleander oligosaccharides have a molecular weight of 2900 Da. The main chain consists of →5)-α-L-Araf-(1→,→3)-α-L-Araf-(1→,→6)-α-D-Glcp-(1→,→4)-α-D-Manp-(1→,→4)-α-D-Glcp-(1→,→4,6)-α-D-Galp-(1→ and α-L-Araf-(1→), and the side chains consist of... β -D-Glc p -(1→Composition).

2. A method for preparing oleander oligosaccharide as described in claim 1, characterized in that: Includes the following steps: S1. Polysaccharide extraction: Cut dried oleander roots into small sections, defatted with 95% ethanol, air-dried, added water, heated to 85 degrees Celsius and extracted for 3 hours, filtered, and the residue was extracted again with an appropriate amount of water. This process was repeated 3-4 times. The filtrates were combined, heated and concentrated to 1 / 10 volume, centrifuged, and 95% ethanol was added to the supernatant until the final ethanol concentration was 70%. The mixture was allowed to stand for 12 hours, and the precipitated polysaccharide was collected and named JZTP70. The supernatant was further concentrated, and anhydrous ethanol was added until the final ethanol concentration was 90%. The mixture was allowed to stand for 12 hours, centrifuged, and the precipitated polysaccharide was collected and named JZTP90. S2. First purification: Dissolve the polysaccharide JZTP90 obtained in step S1 in water, remove the protein and enrich the oligosaccharide using a multi-stage membrane separation system, maintain the temperature of the feed solution at 40-55℃ and the pressure at 0.1-0.6 MPa, retain the enriched oligosaccharide, and freeze-dry to obtain oligosaccharide JZTP-S. S3. Secondary purification: The oligosaccharide JZTP-S obtained in step S2 was subjected to ion exchange column chromatography, and gradient elution was performed sequentially with water and 0.05 M, 0.10 M, 0.15 M, 0.20 M, 0.40 M, 0.8 M, and 2.0 M NaCl until no sugar fraction eluted. At the same time, the elution curve was tracked using the phenol-sulfuric acid method combined with HPGPC, and the sugar fraction was collected according to the elution curve. The sugar fraction in the aqueous fraction was concentrated and freeze-dried to obtain the oleander oligosaccharide in the aqueous fraction, which was named JZTP-W. Then, take an appropriate amount of JZTP-W, dissolve it in water, centrifuge at 8000 rpm, take the supernatant and perform molecular sieve gel column Sephadex S-100 chromatography, elute with water, and simultaneously use the phenol-sulfuric acid method combined with HPGPC to detect the elution curve. According to the elution curve, collect the oligosaccharide fraction with good homogeneity, concentrate and freeze dry to obtain oleander oligosaccharide.

3. The method for preparing oleander oligosaccharides as described in claim 2, characterized in that: In step S1, the weight ratio of dried oleander root to ethanol is 1:6, and the root is soaked for 12 hours for defatting. After defatting, water with a weight of 10 times that of dried oleander root is added for extraction. The amount of water added to the residue is the same as before. Both centrifugations are performed at 10,000 rpm for 30 minutes.

4. The method for preparing oleander oligosaccharides as described in claim 2, characterized in that: In step S2, the polysaccharide JZTP90 is dissolved by adding water until it is completely dissolved. The multi-stage membrane separation system is a three-stage membrane separation system. The first stage uses a ceramic membrane for solution clarification, and the effluent enters the second stage. The second stage uses a 5000 Da ultrafiltration membrane to retain large polysaccharide and protein molecules, and the effluent then enters the third stage nanofiltration. The third stage uses a 500 Da nanofiltration membrane to retain the enriched oligosaccharides. Each stage of filtration is washed 3 to 6 times. Finally, the obtained oligosaccharide components are concentrated under reduced pressure and freeze-dried to obtain JZTP-S. When the material is separated in the multi-stage membrane separation system, the feed temperature is maintained at 50°C and the pressure is 0.5 MPa.

5. The method for preparing oleander oligosaccharides as described in claim 2, characterized in that: In step S3, the material is dissolved in 30 times its weight of water, centrifuged at 8000 rpm for 10 min, concentrated under reduced pressure at a temperature of 40-70℃ and a pressure of 800 Pa, and then freeze-dried at -65℃ and a vacuum of 10-20 Pa.

6. The application of oleander oligosaccharide as described in claim 1, characterized in that: Application of the oleander oligosaccharide in the preparation of immune-enhancing drugs, corresponding tumor immunotherapy drugs, or health products.

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