Gracilaria heteroclada low-molecular-weight polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses a gracilaria variegata low-molecular-weight polysaccharide GBP-1a, the monosaccharide is composed of 82.8% of galactose and 17.2% of glucose through structural analysis, the molecular weight of the uniform polysaccharide is 2887Da, and the polysaccharide has no toxicity to THP-1 mononuclear cells and THP-1 macrophages within a set concentration range through activity detection; and after the polysaccharide is subjected to immune training on THP-1 mononuclear cells and THP-1 macrophages, the immune response of the polysaccharide is obviously higher than that of cells which are not subjected to polysaccharide immune training, so that the training of the polysaccharide is preliminarily deduced to possibly enable the cells to generate acquired immunity similar to vaccines.

Description

Technical Field

[0001] The invention relates to the technical field of polysaccharides, and in particular to a Gracilaria divaricata low molecular weight polysaccharide GBP-1a, a separation and preparation method of the polysaccharide, and an application of the polysaccharide. Background Art

[0002] Gracilaria bailinae is an economic seaweed endemic to the South China Sea. It belongs to the phylum Rhodophyta, class Florideae, orders Gigartinales, family Gracilariaceae, and genus Gracilaria. This species is mainly distributed in the mud bottoms or flowing ditches and stones in the mid-tidal to low-tidal zones in the coastal areas of Guangdong and Hainan provinces. It is an important abalone feed and agar industrial raw material.

[0003] Gracilaria polysaccharides are mainly composed of polygalactose sulfate in structure, and contain small amounts of xylose, glucose, rhamnose and fucose. Its main chain structure is composed of repeating units of 1,3-β-D-galactose and 1,4-α-L-galactose, of which 1,4-L-galactose can be partially or completely replaced by 1,4-3,6-endo-L-galactose. The hydroxyl groups on the main chain often undergo a variety of substitution reactions, including methyl etherification, sulfation, pyruvic acid acetalization, and substitution of monosaccharide residues such as β-D-xylosyl. Due to differences in separation and purification methods, its sulfate content can fluctuate in the range of 0.14-26%. The substitution sites of the sulfate group are mainly distributed at the C-2, C-4 and C-6 positions of 1,3-D-galactose, and the C-2 and C-6 positions of 1,4-L-galactose.

[0004] Gracilaria polysaccharides can be divided into two types: colloidal and non-colloidal. Agar is the main colloidal component, including neutral agarose and acidic sulfur agar. The basic structure of agarose is formed by alternating 1,3-β-D-galactose and 1,4-3,6-ether-L-galactose. Studies have shown that the sulfate content is negatively correlated with the 3,6-ether galactose content. The higher the content of the latter, the greater the gel strength of the agar. Therefore, in industrial production, alkali treatment is often used to increase the 3,6-ether galactose content and reduce the sulfate content to enhance the gel properties of agar. At present, the extraction methods of Gracilaria polysaccharides mainly include:

[0005] 1. Solvent extraction method: Water extraction method: simple operation, small equipment loss, low cost, suitable for industrial production; Acid-base extraction method: can obtain polysaccharide fragments with novel structures, but the equipment is severely corroded.

[0006] 2. Physical-assisted extraction method: high extraction efficiency, convenient separation and purification, but high cost.

[0007] 3. Enzymatic hydrolysis and extraction method: mild conditions, high extraction efficiency, suitable for industrial production, but limited by the lack of specific enzymes.

[0008] Studies have found that colloidal polysaccharides and non-colloidal polysaccharides differ significantly in structural characteristics, solubility properties, and biological activity. Although colloidal polysaccharides have higher dissolution rates and contents, non-colloidal polysaccharides exhibit stronger immune activity. However, existing studies have mostly treated the two as mixed polysaccharides, lacking targeted separation and research. The biological activity of Gracilaria polysaccharides is closely related to their structural characteristics, including the combined effects of multiple factors such as monosaccharide composition and content ratio, sugar residue type and connection mode, molecular weight, and substitution site.

[0009] In summary, although there have been many studies on the extraction process of Gracilaria polysaccharides, there is still a lot of room for further exploration in the separation, structural characterization and structure-activity relationship of colloidal and non-colloidal polysaccharides. Developing more efficient and economical extraction processes and establishing the correlation between polysaccharide structure and biological activity will be important research directions in this field in the future. Summary of the invention

[0010] In order to solve the above technical problems, the technical solution of the present invention is as follows:

[0011] The first aspect of the present invention provides a Gracilaria heteroclada low molecular weight polysaccharide GBP-1a.

[0012] A Gracilaria heteroclada low molecular weight polysaccharide GBP-1a, wherein the monosaccharide composition of the Gracilaria heteroclada low molecular weight polysaccharide is 82.8% galactose and 17.2% glucose in terms of molar percentage, and the structural formula of the Gracilaria heteroclada low molecular weight polysaccharide is as follows:

[0013]

[0014] The molecular weight of the Gracilaria heteroclada low molecular weight polysaccharide is 2887 Da.

[0015] The second aspect of the present invention provides a Gracilaria low molecular weight polysaccharide extract, which contains Gracilaria low molecular weight polysaccharide GBP-1a. It can be understood that the present invention obtains uniform Gracilaria low molecular weight polysaccharide GBP-1a through hot water extraction, macroporous resin separation, membrane ultrafiltration, dialysis bag dialysis, and chromatographic column separation. Therefore, the extract containing Gracilaria low molecular weight polysaccharide GBP-1a in each of the above steps is a Gracilaria low molecular weight polysaccharide extract. At the same time, previous studies have also shown that the Gracilaria extract also contains the following components:

[0016] 1. Agar, content: 20%-30% of dry weight, has the properties of biomedical materials, can be used to prepare culture medium, electrophoresis and chromatography materials, has antiviral activity, can promote gastrointestinal motility, prevent constipation, and prevent the absorption of fat, cholesterol and chemical carcinogens.

[0017] 2. Phycoerythrin is an oligomeric protein that has the effect of photodynamic killing of cancer cells, can be used for pathological antigen detection, and has insulin-like activity.

[0018] 3. Phycocyanin, a photosynthetic antenna pigment protein, has the effect of inhibiting tumor cells, strengthening the immune system, improving hematopoietic function, and can be used as a photosensitizer for laser cancer treatment.

[0019] 4. Polyunsaturated fatty acids, content: accounting for about 50% of the total fatty acids, have the effect of lowering blood pressure and promoting brain development and the improvement of nervous system functions.

[0020] 5. Water-soluble polysaccharides can promote the activity of mouse phagocytes, enhance the activity of immune cells, and have a dose-dependent immunoregulatory effect.

[0021] Therefore, as long as the above-mentioned Gracilaria divaricata low molecular weight polysaccharide extract contains Gracilaria divaricata low molecular weight polysaccharide GBP-1a, and may also contain other active ingredients in the Gracilaria divaricata extraction process, they are all covered within the scope of the Gracilaria divaricata low molecular weight polysaccharide extract.

[0022] The third aspect of the present invention provides a method for extracting low molecular weight polysaccharides from Gracilaria heteroclada, comprising the following steps:

[0023] S1. Water extraction: crush Gracilaria divaricata, mix water and Gracilaria divaricata powder, extract at 85°C-95°C to obtain an extract;

[0024] S2, concentration: filter the extract of step S1, concentrate the filtrate, and let stand at 4°C-10°C to obtain a condensate;

[0025] S3, crude polysaccharide: centrifuge the condensate from step S2, collect the supernatant, pass it through NAK-9 macroporous resin, and use deionized water as the eluent to obtain the crude polysaccharide GBP of Gracilaria isobranchii;

[0026] S4, ultrafiltration: the crude polysaccharide GBP of Gracilaria isobranch obtained in step S3 is passed through a 3500Da ultrafiltration membrane, the upper component of the membrane is taken, dialyzed with a 500Da dialysis bag, the material in the dialysis bag is freeze-dried to obtain Gracilaria isobranch polysaccharide GBP-2, the lower component of the membrane is taken, dialyzed with a 500Da dialysis bag, the material in the dialysis bag is freeze-dried to obtain Gracilaria isobranch polysaccharide GBP-1;

[0027] S5. Redissolve the Gracilaria polysaccharide GBP-1 obtained in step S4 with deionized water, and chromatograph it on a DEAE-52 cellulose column. The eluents are ultrapure water, 0.1M NaCl solution, 0.3M NaCl, 0.6M NaCl, 0.9M NaCl and 1.2M NaCl, respectively, at a flow rate of 1 ml / min. Collect the eluted fractions obtained with ultrapure water, and lyophilize them to obtain the Gracilaria polysaccharide GBP-1a according to claim 1.

[0028] Furthermore, the extraction method further comprises one or more of the following technical features a) to e),

[0029] a) In step S1, the extraction time is 3 hours and the number of extractions is 2 times;

[0030] b) in step S2, the concentration temperature is 65° C.;

[0031] c) in step S3, the centrifugal power is 12000 rpm / min and the centrifugal time is 10 min;

[0032] d) In step S4, the ultrafiltration membrane pressure is 0.4 MPa and the membrane temperature is 40°C;

[0033] e) In step S5, the size of the DEAE-52 cellulose column is 2.6 cm×50 cm.

[0034] The Gracilaria heterobranchii of the present invention is picked from Xuwen County, Guangdong, Qiongzhou Strait, and is washed, dried, and crushed. It can be understood that the Gracilaria heterobranchii low molecular weight polysaccharide extract of the present invention refers to the substance obtained by extracting Gracilaria heterobranchii. The Gracilaria heterobranchii low molecular weight polysaccharide extract of the present invention can be prepared by existing methods, for example: hot water extraction, citric acid extraction, alkali treatment, ultrasonic extraction, microwave extraction and enzyme extraction, etc. As long as the Gracilaria heterobranchii low molecular weight polysaccharide GBP-1a can be obtained, it should not be understood as a limitation of the present invention. In an embodiment of the present invention, the Gracilaria heterobranchii low molecular weight polysaccharide extract is obtained by hot water decoction.

[0035] The fourth aspect of the present invention provides an immunomodulatory drug, comprising the above-mentioned Gracilaria divaricata low molecular weight polysaccharide GBP-1a or the above-mentioned Gracilaria divaricata low molecular weight polysaccharide extract.

[0036] Furthermore, pharmaceutically acceptable excipients are also included.

[0037] Further, the immunomodulatory drug is formulated for oral administration.

[0038] The fifth aspect of the present invention provides a method for training immunity of THP-1 monocytes with low molecular weight polysaccharide GBP-1a from Gracilaria heteroclada, comprising the following steps:

[0039] Step (1) preparing the low molecular weight polysaccharide GBP-1a from Gracilaria isobranchus using 1640 complete culture medium to prepare a GBP-1a working solution;

[0040] Step (2) using GBP-1a working solution directly on THP-1 monocytes, culturing, and washing with a sterile PBS solution to obtain stimulated THP-1 monocytes;

[0041] Step (3) The stimulated THP-1 monocytes are quiescently cultured in an incubator, and subcultured to eliminate the effects of the Gracilaria heteroclada low molecular weight polysaccharide GBP-1a stimulation, thereby obtaining trained THP-1 monocytes;

[0042] Step (4) compares various indicators of trained THP-1 monocytes and untrained THP-1 monocytes after secondary stimulation to obtain the effect of low molecular weight polysaccharide GBP-1a from Gracilaria heteroclada on trained immunity of THP-1 monocytes.

[0043] Furthermore, the culturing in step (2) is for 12 hours;

[0044] In step (3), the static culture is 48 hours, and the subculture is 48 hours;

[0045] The secondary stimulus in step (4) is lipopolysaccharide LPS.

[0046] The sixth aspect of the present invention provides a method for training macrophages to be immune to low molecular weight polysaccharide GBP-1a from Gracilaria heteroclada, comprising the following steps:

[0047] Step (1) preparing the low molecular weight polysaccharide GBP-1a from Gracilaria isobranchus using 1640 complete culture medium to prepare a GBP-1a working solution;

[0048] Step (2) using GBP-1a working solution directly on THP-1 monocytes, culturing, and washing with a sterile PBS solution to obtain stimulated THP-1 monocytes;

[0049] Step (3) stimulating the THP-1 monocytes, quiescently culturing them in an incubator, and inducing them with PMA to induce the THP-1 monocytes into macrophages, thereby obtaining trained macrophages;

[0050] Step (4) compares various indicators of the trained macrophages and untrained macrophages after secondary stimulation to obtain the effect of the low molecular weight polysaccharide GBP-1a of Gracilaria heteroclada on the trained immunity of macrophages.

[0051] Furthermore, the culturing in step (2) is for 12 hours.

[0052] The static culture in step (3) is 48 hours.

[0053] The PMA induction culture in step (3) is 48 hours;

[0054] The secondary stimulus in step (4) is lipopolysaccharide LPS.

[0055] Traditional immunology believes that the immune system is divided into two parts: 1. Innate immune system (also known as natural immunity): It responds quickly but has no memory function, like a "security guard". It immediately handles invaders when it sees them, but does not remember who it has handled, such as macrophages, neutrophils, etc. 2. Adaptive immune system (also known as acquired immunity): It reacts slowly but has immune memory, like a "special police". It remembers invaders that it has handled before and reacts faster and stronger when it encounters them again, such as T cells, B cells, and immune memory produced after vaccination.

[0056] New discovery in 2011: Scientists have discovered that the innate immune system also has a "memory" ability: when innate immune cells (such as macrophages) experience an infection or stimulation, certain persistent changes will occur, which enhance their ability to respond to subsequent infections. This change can last for weeks to months.

[0057] The difference between the two types of immune memory: 1. Adaptive immune memory: highly specific, effective only against specific pathogens. Long duration: can last for several years to a lifetime, and works by producing memory T cells and B cells. 2. Trained immunity (innate immune memory): non-specific: effective against a variety of pathogens, short duration: weeks to months, mainly through epigenetic modification and metabolic reprogramming. This discovery is like discovering that security guards (innate immunity) can not only deal with invaders immediately, but also improve their work ability through "training". Although they don't remember as long as special police (adaptive immunity), they can indeed provide better protection for a certain period of time. This has greatly changed our understanding of the immune system and provided new possibilities for medical treatment.

[0058] In the fifth aspect of the present invention, THP-1 monocytes were trained and immunized by using the low molecular weight polysaccharide GBP-1a of Gracilaria heteroclada, and various indicators of the trained THP-1 monocytes and the untrained THP-1 monocytes after secondary stimulation were compared. It was found that GBP-1a induced THP-1 monocytes to produce "immune memory", and THP-1 monocytes after resting for 4 days and macrophages formed after PMA induction produced a stronger immune response intensity to LPS re-stimulation, but the enhancement of this immune response intensity was not reflected in the expression of all cell transcription and secretion factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1The molecular weight gel permeation chromatogram of the mixed polysaccharide (GBP) of Gracilaria heteroclada after passing through the NAK-9 macroporous resin column and the polysaccharides above the membrane (GBP-2) and below the membrane (GBP-1) after being separated by the ultrafiltration flat membrane 3500Da in the present invention;

[0060] Figure 2 The DEAE Fast Flow elution curve of the Gracilaria heteroclada submembrane component polysaccharide (GBP-1) prepared in the present invention;

[0061] Figure 3 is a high performance gel permeation chromatogram of the molecular weight of the Gracilaria isobranch low molecular weight polysaccharide (GBP-1a) prepared in the present invention;

[0062] Figure 4 The time-of-flight mass spectrum of the Gracilaria heteroclada low molecular weight polysaccharide (GBP-1a) prepared in the present invention;

[0063] Figure 5 The ion chromatogram of the monosaccharide composition of the Gracilaria isobranch low molecular weight polysaccharide (GBP-1a) prepared in the present invention;

[0064] Figure 6 is a Fourier transform infrared spectrum of the Gracilaria heteroclada low molecular weight polysaccharide (GBP-1a) prepared in the present invention;

[0065] Figure 7 The ultraviolet absorption spectrum of the low molecular weight polysaccharide (GBP-1a) of Gracilaria heteroclada obtained in the present invention is shown in FIG.

[0066] Figure 8 This is an analysis chart of methylated monosaccharide residues of the Gracilaria isobranch low molecular weight polysaccharide (GBP-1a) prepared in the present invention;

[0067] Fig. 9 is the nuclear magnetic resonance spectrum of the Gracilaria heteroclada low molecular weight polysaccharide (GBP-1a) prepared in the present invention, a is 1 H spectrum, b is 13 C spectrum, c is COSY spectrum, d is HSQC spectrum, and e is HMBC spectrum;

[0068] Fig.10 The structure diagram of the Gracilaria heteroclada low molecular weight polysaccharide (GBP-1a) prepared in the present invention;

[0069] Fig.11 This is a scanning electron microscope image of the Gracilaria heteroclada low molecular weight polysaccharide (GBP-1a) prepared in the present invention;

[0070] Fig.12 The figure shows the effect of the Gracilaria heteroclada low molecular weight polysaccharide (GBP-1a) prepared in the present invention on the proliferation of THP-1 monocytes (a) and macrophages (b);

[0071] Fig.13 This is a graph showing the effect of the Gracilaria isobranchia low molecular weight polysaccharide (GBP-1a) prepared in the present invention on the cytokine transcription levels (RT-qPCR) of THP-1 monocytes and THP-1 macrophages;

[0072] Fig.14 This is a graph showing the effect of the Gracilaria isobranchia low molecular weight polysaccharide (GBP-1a) prepared in the present invention on the secretion of immune factors by THP-1 monocytes and THP-1 macrophages;

[0073] Fig.15 RT-qPCR was used to evaluate the expression of transcriptional levels of cellular immune factors in THP-1 monocytes and macrophages after training THP-1 monocytes with low molecular weight polysaccharide of Gracilaria heteroclita (GBP-1a);

[0074] Fig.16 ELISA was used to evaluate the expression of cellular immune factors in THP-1 monocytes and macrophages after training THP-1 monocytes with low molecular weight polysaccharide of Gracilaria heteroclada (GBP-1a). DETAILED DESCRIPTION

[0075] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0076] Embodiment 1:

[0077] The preparation of low molecular weight polysaccharide (GBP-1a) from Gracilaria heteroclada comprises the following steps:

[0078] S1. Mixed material extraction: Take naturally dried Gracilaria heteroclada seaweed as raw material, crush it and mix it with distilled water at a solid-liquid ratio of 1:30 (g / mL), stir evenly, and extract at 85°C for 3h, repeat the extraction twice;

[0079] S2. Filtration and concentration: The extract solution obtained in step S1 was filtered through gauze, the filtrate was rotary evaporated to a certain volume at 65°C, and the concentrate was placed in a refrigerator at 4°C overnight;

[0080] S3. The condensed concentrate was centrifuged at 12000 rpm / min for 10 min, the gel part and impurities were discarded, and the supernatant was collected. The obtained supernatant was diluted to 10 times of the original volume, and then the pigment was adsorbed by NAK-9 macroporous resin chromatography column, and the eluent was deionized water. At this time, the obtained Gracilaria isobranch crude polysaccharide (GBP) was obtained;

[0081] S4. Then, the eluate collected in S3 was passed through a 3500Da ultrafiltration membrane (membrane pressure 0.4MPa) to remove impurities and separate the sample solution. The component on the membrane was Gracilaria polysaccharide (GBP-2), and the component below the membrane was Gracilaria polysaccharide (GBP-1). The concentrated solution of Gracilaria crude polysaccharide (GBP), Gracilaria polysaccharide (GBP-2) and Gracilaria polysaccharide (GBP-1) was dialyzed with a 500Da dialysis bag until the conductivity of the dialysate remained constant. The material in the dialysis bag was freeze-dried, and the gel permeation chromatogram of the polysaccharide was shown in Figure 1 The molecular weight determined initially is used as the basis for selecting the molecular weight cutoff of the subsequent ultrafiltration flat membrane. Figure 1 The GBP diagram in the middle is the gel permeation chromatogram of the crude polysaccharide of Gracilaria heteroclada (GBP), the GBP-2 diagram is the gel permeation chromatogram of the crude polysaccharide of Gracilaria heteroclada (GBP-2), and the GBP-1 (solvent is H 2 O) and GBP-1 (solvent is 0.05M NaCl) are gel permeation chromatograms of crude polysaccharide of Gracilaria heteroclada (GBP-1). The purpose of the two figures is to clarify the position of the solvent peak in the gel permeation chromatogram of crude polysaccharide of Gracilaria heteroclada (GBP-1).

[0082] S5. The component polysaccharide (GBP-1) in step S4 was prepared into a solution with deionized water, and chromatographed on a DEAE Fast Flow anion exchange column with a column size of 2.6 cm × 50 cm. The eluents were ultrapure water, 0.1 M NaCl, 0.3 M NaCl, 0.6 M NaCl, 0.9 M NaCl and 1.2 M NaCl, respectively. The flow rate was 1 ml / min, and 10 ml was collected in each tube. The elution curve of the phenol-sulfuric acid method was as follows: Figure 2 The eluate obtained by ultrapure water was collected, freeze-dried, and the above operation was repeated to obtain the low molecular weight polysaccharide of Gracilaria isobranch (GBP-1a).

[0083] Embodiment 2:

[0084] The molecular weight and purity of low molecular weight polysaccharide (GBP-1a) of Gracilaria heteroclada were determined by HPGPC:

[0085] Sample treatment: accurately prepare 0.05M NaCl solution, filter through 0.45μm filter membrane, ultrasonicate for 10min and store at room temperature for later use. Accurately weigh 1 medium Gracilaria isobranch low molecular weight polysaccharide (GBP-1a) and standard, prepare the sample into 5mg / ml solution, centrifuge at 12000rpm for 10min, filter the supernatant with 0.22μm microporous filter membrane, and then transfer the sample to a 1.8ml injection vial;

[0086] Chromatographic column analysis conditions: the chromatographic column is a BRT105-104-102 series gel column (8×300mm); the mobile phase is 0.05M NaCl solution, the flow rate is controlled at 0.6ml / min, the column temperature is 40°C; the injection volume is 20μl; the detector is a differential detector RI-10A.

[0087] Peak molecular weight Mp: The molecular weight of the highest peak. Mp is also an expression of molecular weight distribution, which is used to characterize polymers with extremely narrow molecular weight distribution, such as calibrating polymer standards.

[0088] Number average molecular weight Mn: The number average molecular weight is the statistical average of the molecular weights of all polymer chains in a sample. Mn can be predicted by the polymerization mechanism and determined by measuring the number of molecules in a given mass of sample, such as colligative methods such as end group analysis. If Mn is used to characterize the molecular weight distribution, there are equal numbers of molecules distributed on both sides of Mn.

[0089] Weight average molecular weight Mw: Weight average molecular weight is defined as follows: Compared to Mn, Mw takes into account the contribution of the molecular weight of a single chain to Mw when determining the average molecular weight. The greater the mass of the chain, the greater its contribution to Mw. Mw is determined by a method that is sensitive to the size of the molecules, not just their number. If Mw is used to characterize the molecular weight distribution, then there are equal weights of molecules distributed on both sides of Mw.

[0090] Experimental results: Solvent peak: 2.0min is the peak of 0.05M NaCl; calibration curves of 1gMp-RT (peak molecular weight), 1gMw-RT (weight average molecular weight), and 1gMn-RT (number average molecular weight) were obtained:

[0091] Experimental results: The calibration curves of lgMp-RT (peak molecular weight), lgMw-RT (weight average molecular weight), and lgMn-RT (number average molecular weight) were obtained:

[0092] The equation of the lgMp-RT calibration curve is: y = -0.232x + 11.83, R 2 =0.992;

[0093] The equation of lgMw-RT calibration curve is: y = -0.234x + 11.88, R 2 =0.992;

[0094] The equation of the lgMn-RT calibration curve is: y = -0.234x + 11.87, R 2 =0.992;

[0095] According to the standard curve, the calculation formula was obtained to calculate the molecular weight of each sample. The weight average molecular weight (Mw) of the Gracilaria isobranch low molecular weight polysaccharide (GBP-1a) was 3551Da by HPGPC. The data results are as follows: Figure 3 And as shown in Table 1.

[0096] Table 1 Molecular weight data of low molecular weight polysaccharide (GBP-1a) from Gracilaria heteroclada

[0097]

[0098] Embodiment 2:

[0099] Determination of the specific molecular weight of low molecular weight polysaccharide (GBP-1a) from Gracilaria heteroclada by time-of-flight mass spectrometry

[0100] 10 mg / mL Gracilaria isobranch low molecular weight polysaccharide (GBP-1a) sample aqueous solution was prepared, and the sample solution (1 μL) and 1 M matrix solution (1 μL, acetonitrile: H 2 After mixing with 1 μL of 2% paraformaldehyde (2:1, 1:1), 1 μL was air-dried on a stainless steel target. The primary mass spectrometry of low molecular weight polysaccharides was determined in positive ion mode.

[0101] Experimental results: Figure 4 It shows that the ions produced by GBP-1a are mainly distributed at 2933m / z, and there are very few other components. Combined with the subsequent results, the composition is (Glc) 2 (Gal) 9 (AnGal) 7 SO 3 Na-Na + , that is, the actual molecular weight of the low molecular weight polysaccharide GBP-1a of Gracilaria heteroclada is 2887Da.

[0102] 1. Determination of monosaccharide composition of low molecular weight polysaccharide (GBP-1a) of Gracilaria heteroclada by ion chromatography

[0103] Monosaccharide analysis experimental method: Monosaccharide analysis experimental method: Accurately weigh 5mg±0.05mg of GBP-1a, add 2.5mol / L trifluoroacetic acid (TFA) solution, and heat to hydrolyze at 121℃ for 2h. After concentration and evaporation, add methanol, blow dry with nitrogen, and repeat several times to remove residual TFA; the dried sample is dissolved in sterile deionized water and then tested on the machine. Fucose, aminogalactose hydrochloride, rhamnose, arabinose, glucosamine hydrochloride, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, guluronic acid, glucuronic acid, mannuronic acid standards are mixed to prepare a 10mg / ml standard solution stock solution, diluted 100 times, and then diluted to 1, 5, 10, 20, 30, 40, 50 and 60μg / ml standard solutions for machine detection. According to the single standard method, the concentration of different monosaccharides is determined, and the molar ratio is calculated according to the molar mass of the monosaccharide;

[0104] Machine conditions: Mobile phase A: ddH 2 O, B phase: 200mM NaOH, C phase: 200mM NaOH and 500mM NaAC; separation column: Dionex TM CarboPac TM PA20 (150 mm × 3 mm, 6.5 μm), flow rate 0.5 ml / min. Gradient elution program: 0-25 min, 97.5% A, 2.5% B; 25-25.1 min, 97.5% A linearly changed to 77.5%, 0% C linearly changed to 20%; 25.1-40 min, 77.5% A, 2.5% B, 20% C; 40-40.1 min, C linearly changed to 100%; 40.1-50 min, 100% C; 50-50.1 min, 0% A linearly changed to 97.5%, 0% B linearly changed to 2.5%, 50.1-60 min, 97.5% A, 2.5% B;

[0105] Mixed standard solvent peak: 2.0min is the peak of sodium hydroxide, 40min is the peak of sodium acetate;

[0106] Experimental results: Ion chromatography analysis results are as follows Figure 5 As shown in Table 2, compared with the monosaccharide standard sample, it was found that the Gracilaria isobranch low molecular weight polysaccharide (GBP-1a) in the present invention is composed of galactose (82.8%) and glucose (17.2%).

[0107] Table 2 Monosaccharide composition of low molecular weight polysaccharide (GBP-1a) of Gracilaria heteroclita

[0108]

[0109] 2. The functional groups and protein nucleic acid contents of the low molecular weight polysaccharide (GBP-1a) of Gracilaria heteroclita in Example 1 were determined by Fourier transform infrared spectrometer and UV-visible photometer, respectively.

[0110] Experimental method: Weigh 1 mg of fully dried GBP-1a sample, mix and grind it with 100 mg of dry potassium bromide under a heating lamp, press it into transparent tablets in a tablet press, and then place the tablets on a VERTEX 70 infrared spectrometer at 4000-400 cm -1 Infrared data were collected within the range; GBP-1a was prepared into a 500 μg / ml solution dissolved in ultrapure water, ultrapure water was used as a blank control group, and scanned at the full wavelength of 190-600 nm using a UV-visible spectrophotometer.

[0111] Experimental results: The infrared spectrum and ultraviolet spectrum of low molecular weight polysaccharide (GBP-1a) of Gracilaria heteroclada are as follows: Figure 6 and Figure 7 As shown. The infrared spectrum shows that at 3413.84 and 2922.02 cm -1 The absorption peaks observed near 1157 and 1074 cm are important characteristic absorption peaks of carbohydrates, corresponding to the stretching vibration of intermolecular or intramolecular OH bonds and the CH of alkyl groups, respectively. -1 The absorption peaks at 1411.83, 1251.74, 869.85 and 931.57 cm -1 ) are respectively related to the asymmetric stretching vibration of the sulfate group (O=S=O), the symmetric stretching vibration of COS and the COC of 3,6-ether galactose, indicating that GBP-1a contains sulfate groups and 3,6-ether galactose. Figure 7 The 740.43 and 771.49 cm-1 caused by the backbone bending of the galactose ring are also shown. -1 The peaks at 893.00 cm-1 and 893.00 cm-2 associated with the CH bending of the isomeric carbons in the β-galactose residues -1 The results of UV full wavelength scanning showed that there were no obvious absorption peaks at 260nm and 280nm, indicating that GBP-1a did not contain nucleic acids and proteins, indicating that the purification effect of GBP-1a was good.

[0112] 3. Example 1: After methylation, hydrolysis and acetylation, the low molecular weight polysaccharide (GBP-1a) of Gracilaria heteroclita was measured by GC-MS and compared with the standard mass spectrum library

[0113] Experimental method: Weigh the low molecular weight polysaccharide (GBP-1a) sample (2-3 mg) of Example 1 and place it in a glass reaction bottle, add 1 ml of anhydrous DMSO, quickly add methylation reagent A solution, seal, dissolve under ultrasound, and then add methylation reagent B solution. React in a magnetic stirring water bath at 30°C for 60 minutes. Finally, add 2 ml of ultrapure water to the above mixture to terminate the methylation reaction. Take the methylated polysaccharide, add 1 ml of 2M trifluoroacetic acid (TFA) to hydrolyze for 90 minutes, and evaporate to dryness on a rotary evaporator. Add 2 ml of double distilled water to the residue, reduce 60 mg of sodium borohydride for 8 hours, add glacial acetic acid to neutralize, rotary evaporate, dry in an oven at 101°C, then add 1 ml of acetic anhydride to acetylate at 100°C for 1 hour, and cool. Then add 3 ml of toluene, concentrate and evaporate to dryness under reduced pressure, and repeat 4-5 times to remove excess acetic anhydride. The acetylated product is washed with 3 ml of CH 2 Cl 2 After dissolving, transfer to a separatory funnel, add a small amount of distilled water and shake thoroughly, then remove the upper aqueous solution, and repeat this process 4 times. 2 Cl 2 The layer was dried with an appropriate amount of anhydrous sodium sulfate, fixed to 10 ml, and placed in a liquid phase vial;

[0114] Analytical instrument: Acetylation product samples were determined using Shimadzu GCMS-QP 2010 gas chromatography-mass spectrometry;

[0115] GC-MS conditions: RXI-5SIL MS column 30m*0.25mm*0.25μm, programmed temperature conditions: starting temperature 120℃, heating at 3℃ / min to 250℃ / min, maintaining for 5min, injection port temperature 250℃, detector temperature 250℃ / min, carrier gas helium, flow rate 1ml / min;

[0116] Experimental results: The methylated monosaccharide forms of low molecular weight polysaccharide (GBP-1a) of Gracilaria heteroclita Figure 8As shown in Table 3, the sugar residues and proportions were analyzed by methylation treatment and GC-MS. The results showed that there were 7 monosaccharide residue linkage modes in the low molecular weight polysaccharide (GBP-1a) of Gracilaria heteroclita, namely 2,3,4,6-Me4-Galp, 2,3,6-Me3-Galp, 2,3,6-Me3-Glcp, 2,4,6-Me3-Galp, 2,3,4-Me3-Glcp, 2,3-Me2-Galp, and 2,4-Me2-Galp. These 7 monosaccharide residues are all related to galactose and glucose, which once again proves that GBP-1a is composed of galactose and glucose. In addition, based on the absorption peaks of 3,6-ether galactose and sulfate group in the infrared spectrum results, combined with the proportion of branched sugar content in the methylation results, it is inferred that 2,3-Me2-Galp and 2,4-Me2-Galp are caused by the presence of 3,6-ether galactose and sulfate group at position 6, respectively, and the specific analysis is carried out in combination with the one-dimensional and two-dimensional NMR spectrum results.

[0117] Table 3 Analysis of results of methylated polysaccharide acetyl ester (PMAA) of Gracilaria isobranchii low molecular weight polysaccharide (GBP-1a)

[0118]

[0119] 4. The structure of the low molecular weight polysaccharide (GBP-1a) of Gracilaria heteroclada in Example 1 was inferred by superconducting nuclear magnetic resonance spectrometer

[0120] Experimental method: Weigh about 40 mg of GBP-1a sample and dissolve it in 0.55 ml of D 2 O, placed in a water bath to fully dissolve, then centrifuged to take the supernatant and put it into a nuclear magnetic resonance tube, and performed one-dimensional ( 1 H NMR and 13 CNMR) and two-dimensional (COSY, HSQC and HMBC) spectra;

[0121] Experimental results: The NMR results of low molecular weight polysaccharide (GBP-1a) of Gracilaria heteroclada are as follows Fig. 9 As shown, based on the results of monosaccharide composition and methylation analysis, the structure of GBP-1a was studied by one-dimensional and two-dimensional NMR analysis. Fig. 9 a and Fig. 9 b respectively gives 1 H NMR and 13 One-dimensional spectrum including CNMR. At δ3.5-5.5ppm ( 1 H NMR) and δ 60-110 ppm ( 13Most of the spectral signals in GBP-1a observed in the C NMR range are considered to be typical NMR signals of polysaccharides. In the hetero-head region, seven significant hetero-head proton coupling signals were found at δ4.60, δ5.26, δ5.24, δ4.58, δ5.14, δ5.15 and δ4.48 ppm. By examining 13 The cross peaks in the heterogenic region of C NMR and HSQC spectra ( Fig. 9 d), the heterologous head carbon signals corresponding to the above heterologous head protons were determined to be δ96.09, δ92.37, δ100.96, δ102.00, δ97.92, δ98.13 and δ103.05 ppm respectively. This observation indicates the presence of 7 glycosyl residues, and these residues are classified as A, B, C, D, E, F and G according to the intensity of their chemical shift signals. Then, according to the COSY spectrum ( Fig. 9 c), find out H1-H6 of the 7 sugar residues and their corresponding C signals in the HSQC spectrum. Finally, combined with the HMBC spectrum ( Fig. 9 e) Determine the connection mode of the seven monosaccharide residues and obtain the structure of GBP-1a. It is speculated that the structure of GBP-1a is as follows Fig.10 shown.

[0122] Its main chain structure is:

[0123] α-D-Galp-1→3-β-D-Galp6S-1→4-α-L-AnGalp-1→4-α-D-Glcp-1→[6-α-D-Glcp-1→4-α-L-AnGalp-1] 6 →4-α / β-D-Galp. “6S” represents the sulfate group linked to the C6 position, and “AnGalp” represents 3,6-endogalactose.

[0124] Table 4 Chemical shift assignments of monosaccharide residues in Gracilaria isobranchia low molecular weight polysaccharide (GBP-1a)

[0125]

[0126] 5. Example 1 Scanning electron microscopy of Gracilaria heteroclada low molecular weight polysaccharide (GBP-1a)

[0127] Experimental method: Take about 5 mg of the dried low molecular weight polysaccharide of Gracilaria heteroclita (GBP-1a) of Example 1, adhere it to a conductive carbon film containing a double-sided adhesive, place it in the sample chamber of an MC1000 ion sputtering instrument (HITACHI, Japan), and spray gold for about 40 seconds. After the sample is taken out, place it in a SU8100 scanning electron microscope (HITACHI, Japan) for observation, and the acceleration voltage is set to 2KV.

[0128] Experimental results: The scanning results of low molecular weight polysaccharide (GBP-1a) of Gracilaria heteroclita under electron microscope, such as Fig.11 As shown, under electron microscopes of different magnifications, the samples all exhibit filamentous structures with varying degrees of thickness.

[0129] Embodiment 3:

[0130] 6. Example 1 Determination of the immunomodulatory activity and trained immune induction effect of low molecular weight polysaccharide (GBP-1a) of Gracilaria heteroclita

[0131] S1. Culture of THP-1 monocytes and THP-1 macrophages

[0132] Experimental content: Human monocytic leukemia THP-1 cells were cultured in RPMI 1640 medium containing 10% FBS and 1% (v / v) double antibody at 37°C and 5% CO 2 THP-1 monocytes can be induced into THP-1 macrophages by treating them with PMA at a final concentration of 100 ng / mL for 24 hours and then leaving them in complete medium for 24 hours. When the cells adhere to the wall and almost cover the bottom of the culture flask, they can be passaged or plated for the next experiment.

[0133] recovery:

[0134] Take out the cells from the -80℃ refrigerator or liquid nitrogen tank, use a 37℃ water bath to completely thaw the cell suspension within 1 minute, transfer the thawed cell suspension to a centrifuge tube containing complete culture medium and mix the cell suspension, centrifuge at 1000rpm / min for 3 minutes, discard the culture medium, add 1mL of complete culture medium to the cell pellet, mix the cell pellet thoroughly with a pipette and transfer it to a cell culture flask that has been added with complete culture medium, gently shake the culture flask to evenly distribute the cells, and then transfer it to an incubator for culture.

[0135] Passaging:

[0136] Before induction, THP-1 monocytes were suspension cells. When the cell density reached 8×10 5 When the density is about 1×10 cells / mL, the cell suspension can be aspirated, centrifuged and transferred to a new culture bottle containing complete culture medium for culture. The minimum density for subculturing should be no less than 1×10 5 cells / mL.

[0137] Cryopreservation:

[0138] The cell suspension was collected and centrifuged at 1000 rpm / min for 3 min. After removing the supernatant, an appropriate amount of cell freezing solution was added to the cell pellet, which was evenly pipetted and transferred to a sterile cryopreservation tube. The temperature was gradually reduced to -80°C and finally stored in a liquid nitrogen tank.

[0139] train:

[0140] GBP-1a working solution was prepared using 1640 complete culture medium. First, THP-1 monocytes were cultured at 5 × 10 5 The cells were plated in a 6-well plate at a density of 100 μg / ml and GBP-1a working solution was immediately added to make the final concentration 100 μg / ml. The cells were incubated at 37°C and 5% CO 2 Incubate in a culture incubator for 12 h. Then, wash the cells twice with warm sterile PBS solution, resuspend them in a T25 cell flask, and culture them in a culture incubator for 4 days (one subculture in the middle). After the rest period, resuspend and count the trained THP-1 monocytes, and adjust the cell density to 5×10 5 / ml, plated in a 6-well plate, added with LPS solution (1μg / ml) for 12h, and then collected the samples of each group.

[0141] S2. Effects of low molecular weight polysaccharide from Gracilaria heteroclada (GBP-1a) on THP-1 monocytes and THP-1 macrophages

[0142] Experimental content: The CCK-8 method was used to detect the effect of low molecular weight polysaccharide of Gracilaria heteroclada (GBP-1a) on the proliferation of THP-1 monocytes and THP-1 macrophages, the RT-qPCR method was used to detect the effect on the expression of immune factor genes (TNF-α, IL-6, IL-1β), and the ELISA method was used to detect the effect on the expression of immunoregulatory factors (TNF-α, IL-6, IL-1β), to evaluate its immunoregulatory activity and training immune effect.

[0143] Experimental operation:

[0144] 1. Effect on the proliferation of THP-1 monocytes and THP-1 macrophages: CCK-8 kit was used to determine the effect of low molecular weight polysaccharide of Gracilaria isobranch (GBP-1a) on the proliferation of THP-1 monocytes and THP-1 macrophages. THP-1 monocytes: The cells in the culture flask were blown down with complete culture medium, and the density of THP-1 monocytes was adjusted to 3.0×10 5 / mL, and then add the working solution of Gracilaria isobranch low molecular weight polysaccharide (GBP-1a) to make its concentration 25, 50, 100, 200, 400 and 800 μg / mL, respectively. After mixing, add 100 μL to each well of the 96-well plate and culture in an incubator for 24 h.

[0145] THP-1 macrophages: The cells in the culture flask were blown down with complete culture medium, and the density of THP-1 monocytes was adjusted to 3.0×10 5 / mL, add PMA working solution to make the concentration of 100ng / ml, after 24h, THP-1 changes from suspension state to adherent state, discard the supernatant culture medium, add new complete culture medium and let it stand for 24h. Then discard the supernatant, add low molecular weight polysaccharide of Gracilaria isobranch (GBP-1a) working solution to make its concentration 25, 50, 100, 200, 400 and 800μg / mL, set up blank control group and LPS control group at the same time, add cells and complete culture medium to the blank control group, the LPS group is added with a concentration of 1μg / mL, and set up 5 duplicate wells for each group. After culturing in an incubator for 24h, add 10μL CCK-8 reagent to each well, incubate in dark for 1h, and detect and record the absorbance at a wavelength of 450nm with an enzyme marker.

[0146] 2. Effects on the gene expression of immune factors in THP-1 monocytes and THP-1 macrophages: RT-qPCR was used to evaluate the effects of GBP-1a on the transcriptional levels of TNF-1α, IL-6, and IL-1β genes in THP-1 monocytes and THP-1 macrophages. (1) Seed plate: THP-1 monocytes and THP-1 macrophages were plated at 5.0 × 10 5 The cells were inoculated at a density of 100 μg / ml (see ① for specific operations); (2) Drug addition: GBP-1a concentrations of 50, 100, and 200 μg / mL were added to complete culture medium and treated in an incubator for 12 h; (3) RNA extraction, reverse transcription, and Real-Time PCR were performed in sequence, and the relative quantitative method was used to calculate the gene expression multiple, with β-Actin as the internal reference. The results were expressed as relative expression levels, and the formula was 2 -ΔΔCt .

[0147] 3. Effect on the release of immune factors by THP-1 monocytes and THP-1 macrophages: ELISA kits were used to determine the effect of GBP-1a treatment for 12 hours on the release of cytokines by THP-1 monocytes and THP-1 macrophages. The specific operation was the same as ②, only the corresponding supernatant was collected, and the secretion of the corresponding immune factors was detected using TNF-α, IL-6, and IL-1β kits.

[0148] Experimental results:

[0149] 1. The present invention uses CCK-8 method to detect the effect of low molecular weight polysaccharide of Gracilaria heteroclidae (GBP-1a) on the proliferation of THP-1 monocytes and THP-1 macrophages. The results are as follows Fig.12As shown, GBP-1a can promote the proliferation of THP-1 monocytes and THP-1 macrophages at various concentrations, indicating that the low molecular weight polysaccharide of Gracilaria heteroclada (GBP-1a) has no cytotoxicity to THP-1 monocytes and THP-1 macrophages within the concentration range of the set group, and the promoting effect of lower concentrations is more significant than that of higher concentrations.

[0150] 2. If Fig.13 As shown in the figure, in THP-1 monocytes and THP-1 macrophages, at doses of 50, 100, and 200 μg / mL of GBP-1a, the transcription levels of immune regulation-related cytokines IL-6 and IL-1β were upregulated to varying degrees with the increase of GBP-1a concentration. There was no obvious gradient relationship between the transcription level of TNF-α and the concentration, but it was significantly increased compared with the blank control group.

[0151] 3. The present invention uses an ELISA kit to detect the effect of low molecular weight polysaccharide of Gracilaria heteroclita (GBP-1a) on the release of immune factors by THP-1 monocytes and THP-1 macrophages, indicating that low molecular weight polysaccharide of Gracilaria heteroclita (GBP-1a) promotes their immune response. Fig.14 As shown in the figure, compared with the blank control group, the secretion of cellular immune factors (TNF-α, IL-6, IL-1β) increased in a dose-dependent manner under the treatment of GBP-1a at doses of 50, 100 and 200 μg / mL. The above results indicate that low molecular weight polysaccharide of Gracilaria heteroclita (GBP-1a) can promote the release of immune-related factors in THP-1 monocytes and THP-1 macrophages.

[0152] 4. If Fig.15 , 16 As shown, where a is, (G+L) is the stimulation of THP-1 monocytes with low molecular weight polysaccharides (GBP-1a) of Gracilaria heteroclada for 12 hours, washed, cultured statically for 48 hours, subcultured for 48 hours, and tested after secondary stimulation with LPS polysaccharide for 12 hours. (C+L) is the direct stimulation of THP-1 monocytes with LPS polysaccharide for 12 hours and then tested. (G+C) is the stimulation of THP-1 monocytes with low molecular weight polysaccharides (GBP-1a) of Gracilaria heteroclada for 12 hours, washed, cultured statically for 48 hours, subcultured for 48 hours, and tested after secondary stimulation with LPS polysaccharide for 12 hours. (C+C) is the result of direct testing of THP-1 monocytes (quality control group without polysaccharide and LPS in Example 1).

[0153] Wherein b is, (GP+L) is THP-1 monocytes stimulated by low molecular weight polysaccharide (GBP-1a) of Gracilaria heteroclada for 12 hours, washed, cultured in quiescent state for 48 hours, cultured in PMA for 48 hours, and tested after secondary stimulation with LPS polysaccharide for 12 hours. (CP+L) is THP-1 monocytes, cultured in PMA for 48 hours, and directly stimulated with LPS polysaccharide for 12 hours, and tested. (GP+C) is THP-1 monocytes stimulated by low molecular weight polysaccharide (GBP-1a) of Gracilaria heteroclada for 12 hours, washed, cultured in quiescent state for 48 hours, cultured in PMA for 48 hours, and tested after secondary stimulation with LPS polysaccharide for 12 hours without LPS polysaccharide. (CP+C) is THP-1 monocytes, cultured in PMA for 48 hours, and directly tested (quality control group without polysaccharide and LPS in Example 1).

[0154] After 12 hours of LPS re-stimulation of GBP-1a-trained THP-1 monocytes (G+L) and untrained cells (C+L), the transcription levels of cytokines, IL-6, and IL-1β and cell secretion factors were significantly increased. The transcription level of TNF-α and cytokines were either increased or decreased after LPS secondary stimulation. "G+C" in the figure indicates the state of no stimulation after 4 days of rest after training. Based on this, it is speculated that GBP-1a induces THP-1 monocytes to produce "immune memory". After 4 days of rest, THP-1 monocytes and macrophages formed after PMA induction have a stronger immune response to LPS re-stimulation, but this enhancement of the intensity of the immune response is not reflected in the expression of all cell transcription and secretion factors.

[0155] The above is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited to the above content. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A low molecular weight polysaccharide GBP-1a from Gracilaria heteroclada, characterized in that: In terms of molar percentage, the monosaccharide composition of the Gracilaria isobranch low molecular weight polysaccharide is 82.8% galactose and 17.2% glucose. The structural formula of the Gracilaria isobranch low molecular weight polysaccharide is as follows: The molecular weight of the Gracilaria heteroclada low molecular weight polysaccharide is 2887 Da.

2. A low molecular weight polysaccharide extract from Gracilaria heteroclada, characterized in that: The Gracilaria heteroclada low molecular weight polysaccharide extract comprises the Gracilaria heteroclada low molecular weight polysaccharide GBP-1a described in claim 1.

3. A method for extracting low molecular weight polysaccharides from Gracilaria heteroclada, characterized in that: The following steps are included: S1. Water extraction: crush Gracilaria divaricata, mix water and Gracilaria divaricata powder, extract at 85°C-95°C to obtain an extract; S2, concentration: filter the extract of step S1, concentrate the filtrate, and let stand at 4°C-10°C to obtain a condensate; S3, crude polysaccharide: centrifuge the condensate from step S2, collect the supernatant, pass it through NAK-9 macroporous resin, and use deionized water as the eluent to obtain the crude polysaccharide GBP of Gracilaria isobranchii; S4, ultrafiltration: the crude polysaccharide GBP of Gracilaria isobranch obtained in step S3 is passed through a 3500Da ultrafiltration membrane, the upper component of the membrane is taken, dialyzed with a 500Da dialysis bag, the material in the dialysis bag is freeze-dried to obtain Gracilaria isobranch polysaccharide GBP-2, the lower component of the membrane is taken, dialyzed with a 500Da dialysis bag, the material in the dialysis bag is freeze-dried to obtain Gracilaria isobranch polysaccharide GBP-1; S5. Redissolve the Gracilaria polysaccharide GBP-1 obtained in step S4 with deionized water, and chromatograph it on a DEAE-52 cellulose column. The eluents are ultrapure water, 0.1M NaCl solution, 0.3M NaCl, 0.6M NaCl, 0.9M NaCl and 1.2M NaCl, respectively, at a flow rate of 1 ml / min. Collect the eluted fractions obtained with ultrapure water, and lyophilize them to obtain the Gracilaria polysaccharide GBP-1a according to claim 1.

4. The extraction method according to claim 3, characterized in that: Contains any one or more of the following technical features a)-e), a) In step S1, the extraction time is 3 hours and the number of extractions is 2 times; b) in step S2, the concentration temperature is 65° C.; c) in step S3, the centrifugal power is 12000 rpm / min and the centrifugal time is 10 min; d) In step S4, the ultrafiltration membrane pressure is 0.4 MPa and the membrane temperature is 40°C; e) In step S5, the size of the DEAE-52 cellulose column is 2.6 cm×50 cm.

5. An immunomodulatory drug, characterized in that: It comprises the Gracilaria heteroclada low molecular weight polysaccharide GBP-1a described in claim 1 or the Gracilaria heteroclada low molecular weight polysaccharide extract described in claim 2.

6. The immunomodulatory drug according to claim 5, characterized in that: Also included are pharmaceutically acceptable excipients.

7. A method for training THP-1 monocytes to be immune using low molecular weight polysaccharide GBP-1a from Gracilaria heteroclada, characterized in that: The steps include: Step (1) preparing the low molecular weight polysaccharide GBP-1a from Gracilaria isobranchus using 1640 complete culture medium to prepare a GBP-1a working solution; Step (2) using GBP-1a working solution directly on THP-1 monocytes, culturing, and washing with a sterile PBS solution to obtain stimulated THP-1 monocytes; Step (3) The stimulated THP-1 monocytes are quiescently cultured in an incubator, and subcultured to eliminate the effects of the low molecular weight polysaccharide GBP-1a of Gracilaria heteroclita to obtain trained THP-1 monocytes; Step (4) compares various indicators of trained THP-1 monocytes and untrained THP-1 monocytes after secondary stimulation to obtain the effect of low molecular weight polysaccharide GBP-1a from Gracilaria heteroclada on trained immunity of THP-1 monocytes.

8. A method for training macrophages to be immune using low molecular weight polysaccharide GBP-1a from Gracilaria heteroclada, characterized in that: The steps include: Step (1) preparing the low molecular weight polysaccharide GBP-1a from Gracilaria isobranchus using 1640 complete culture medium to prepare a GBP-1a working solution; Step (2) using GBP-1a working solution directly on THP-1 monocytes, culturing, and washing with a sterile PBS solution to obtain stimulated THP-1 monocytes; Step (3) stimulating the THP-1 monocytes, quiescently culturing them in an incubator, and inducing them with PMA to induce the THP-1 monocytes into macrophages, thereby obtaining trained macrophages; Step (4) compares various indicators of the trained macrophages and untrained macrophages after secondary stimulation to obtain the effect of the low molecular weight polysaccharide GBP-1a of Gracilaria heteroclada on the trained immunity of macrophages.

9. The method for training THP-1 monocytes to be immuned by low molecular weight polysaccharide GBP-1a from Gracilaria heteroclada according to claim 7, characterized in that: The culturing in step (2) is 12 hours; In step (3), the static culture is 48 hours, and the subculture is 48 hours; The secondary stimulus in step (4) is lipopolysaccharide LPS.

10. The method for training macrophages to be immune using low molecular weight polysaccharide GBP-1a from Gracilaria heteroclada according to claim 8, characterized in that: The culturing in step (2) is 12 hours, The static culture in step (3) is 48 hours. The PMA induction culture in step (3) is 48 hours; The secondary stimulus in step (4) is lipopolysaccharide LPS.

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