阅读说明：本技术 一种多糖sm-0.4m及其制备的抗肿瘤产品 (Polysaccharide SM-0.4M and anti-tumor product prepared from same ) 是由 杨永晶 田文慧 尹星星 王学红 魏涛 于 2020-12-09 设计创作，主要内容包括：本发明公开了一种多糖SM-0.4M,是从树莓多糖中分离得到,并鉴定出其具体的糖链结构,通过试验验证多糖SM-0.4M可促进巨噬细胞增殖,并可促进巨噬细胞释放NO、上调TNF-α和IL-6表达,进而增强巨噬细胞的吞噬作用,提高免疫力,还有利于巨噬细胞发挥抗肿瘤的作用,可用于制备相关产品,同时为进一步探明树莓多糖的构效关系及开发多糖类抗肿瘤药物和免疫佐剂提供参考。(The invention discloses a polysaccharide SM-0.4M, which is separated from raspberry polysaccharide, a specific sugar chain structure of the polysaccharide is identified, experiments verify that the polysaccharide SM-0.4M can promote macrophage proliferation, promote the macrophage to release NO, and up-regulate TNF-alpha and IL-6 expression, further enhance phagocytosis of the macrophage, improve immunity, and is beneficial to the macrophage to play an anti-tumor role, so that the polysaccharide SM-0.4M can be used for preparing related products, and simultaneously provides references for further exploring the structure-activity relationship of the raspberry polysaccharide and developing polysaccharide anti-tumor drugs and immune adjuvants.)

1. A polysaccharide SM-0.4M, characterized in that the sugar chain structure is as follows:

2. polysaccharide SM-0.4M according to claim 1, characterised in that the weight average molecular weight Mw is 86000 to 91000, preferably 88997; the number average molecular weight Mn is 57000-62000, preferably 59284; further, the peak molecular weight Mp is 67000 to 72000, preferably 70366.

3. The process for the preparation of the polysaccharide SM-0.4M according to claim 1, characterized in that the raspberry polysaccharide is subjected to ion exchange chromatography with DEAE Sepharose Fast Flow, after elution of the first fraction with water, the second fraction is eluted with an aqueous solution of NaCl from 0.15mol/L to 0.25mol/L and the third fraction is eluted with an aqueous solution of NaCl from 0.35mol/L to 0.45mol/L to give the polysaccharide SM-0.4M; further, the eluent for eluting the second component is 0.2mol/L of NaCl aqueous solution, and the eluent for eluting the third component is 0.4mol/L of NaCl aqueous solution;

further, the amount of water is 4-6 column volumes, preferably 5 column volumes;

further, the using amount of the NaCl aqueous solution of 0.15-0.25 mol/L is 4-6 column volumes, preferably 5 column volumes;

furthermore, the using amount of the NaCl aqueous solution of 0.35-0.45 mol/L is 4-6 column volumes, preferably 5 column volumes.

4. The preparation method of claim 3, wherein the extraction method of raspberry polysaccharide comprises: defatting Rubi fructus, removing monosaccharide and pigment, extracting the obtained solid with water, and precipitating with ethanol;

further, the amount of water used in water extraction is 5-30 times of the solid matter, and after extraction, the water extraction solution is concentrated to 0.1-0.5 time, preferably 0.25 time of the original volume; then mixing the mixture with 3-5 times of ethanol with the volume fraction of 85-95%, preferably ethanol with the volume fraction of 95%;

further, the water extraction condition is ultrasonic extraction;

further, the power of ultrasonic extraction is 50-70W, and the temperature is 50-90 ℃;

furthermore, the time of ultrasonic extraction is 60min to 100 min.

5. Use of the polysaccharide SM-0.4M according to claim 1 or 2 for the preparation of a product for enhancing immunity.

6. Use according to claim 5, characterized in that the product is an immunological adjuvant.

7. Use of the polysaccharide SM-0.4M according to claim 1 or 2 for the preparation of a product for promoting macrophage proliferation or/and enhancing macrophage phagocytosis.

8. The use according to claim 7, wherein the product is one or more of an NO-releasing agent, a TNF- α agonist, an IL-6 agonist.

9. Use according to any one of claims 5 to 8, wherein the product is a product for the prevention and/or treatment of tumours.

10. Use of the polysaccharide SM-0.4M according to claim 1 or 2 for the preparation of a product for the prevention and/or treatment of tumors.

Technical Field

The invention belongs to the technical field of natural products, and particularly relates to polysaccharide SM-0.4M in raspberry polysaccharide, a preparation method and application thereof.

Background

Plant polysaccharides, also called plant polysaccharides, are polysaccharides with a polymerization degree of more than 10 produced by plant cell metabolism, are natural polymer polymers formed by connecting aldose or ketose through glycosidic bonds, are important biological macromolecules in organisms, and are one of basic substances for maintaining normal operation of life activities.

Meanwhile, the plant polysaccharide is a natural macromolecule with various well-known biological activities, and most of the plant polysaccharide has remarkable immunoregulation activity and in-vivo anti-tumor activity. They can not only directly affect the functions of multiple immune cells such as macrophage, T lymphocyte, B lymphocyte, natural killer cell, etc., but also regulate the production of related cell factor and complement, effectively increase the relation between immune cells and regulate the immune system from multiple aspects and layers. In addition to its safety and non-toxicity characteristics, plant polysaccharides are ideal substances for developing antitumor immunotherapeutic drugs or immunoadjuvants.

Researches show that the polysaccharide derived from the fruit of the red raspberry introduced from the Qinghai-Tibet plateau has obvious in-vivo anti-tumor immune activity and the synergistic effect of chemotherapeutic drugs. In a tumor-bearing mouse model (malignant melanoma), the raspberry crude polysaccharide has very obvious tumor inhibition activity (the tumor inhibition rate of 400mg/kg raspberry polysaccharide is up to 59.95%, and the tumor inhibition rate of a positive control docetaxel injection is 66.49%), has no obvious influence on the weight of a tumor-bearing mouse, and has no damage to the liver and the kidney of the tumor-bearing mouse.

However, the specific components in raspberry polysaccharide are not deeply researched, and if sugar chain structures of different components in raspberry polysaccharide can be clearly defined, the method has important significance for development of antitumor drugs and immune adjuvants of the polysaccharide.

Disclosure of Invention

The invention mainly solves the technical problem of providing polysaccharide SM-0.4M which can improve immunity.

The invention provides polysaccharide SM-0.4M, wherein a sugar chain structure of the polysaccharide is as follows:

further, the polysaccharide SM-0.4M has a weight average molecular weight Mw of 86000-91000, preferably 88997; the number average molecular weight Mn is 57000-62000, preferably 59284.

Further, the polysaccharide SM-0.4M has a peak molecular weight Mp of 67000 to 72000, preferably 70366.

The invention also provides a preparation method of the polysaccharide SM-0.4M, which comprises the following steps: performing ion exchange chromatography on raspberry polysaccharide by using DEAE Sepharose Fast Flow, eluting a first component by using water, eluting a second component by using 0.15-0.25 mol/L NaCl aqueous solution, and eluting a third component by using 0.35-0.45 mol/L NaCl aqueous solution to obtain polysaccharide SM-0.4M.

Further, the eluent for eluting the second component is 0.2mol/L NaCl aqueous solution, and the eluent for eluting the third component is 0.4mol/L NaCl aqueous solution.

Further, the amount of water used is 4-6 column volumes, preferably 5 column volumes.

Furthermore, the amount of the NaCl aqueous solution of 0.15mol/L to 0.25mol/L is 4 to 6 column volumes, preferably 5 column volumes.

Furthermore, the using amount of the NaCl aqueous solution of 0.35-0.45 mol/L is 4-6 column volumes, preferably 5 column volumes.

Raspberry polysaccharides have been extensively studied in the art, and in the art, raspberry polysaccharides prepared by different methods can be used in the present invention.

In a specific embodiment of the present invention, the extraction method of raspberry polysaccharide comprises: defatting Rubi fructus, removing monosaccharide and pigment, extracting the obtained solid with water, and precipitating with ethanol.

Further, the amount of water used in water extraction is 5-30 times of the solid matter, and after extraction, the water extraction solution is concentrated to 0.1-0.5 time, preferably 0.25 time of the original volume; and then mixing with 85-95% ethanol with 3-5 times volume of volume, preferably 95% ethanol.

Further, the water extraction condition is ultrasonic extraction.

Further, the power of ultrasonic extraction is 50-70W, and the temperature is 50-90 ℃.

Furthermore, the time of ultrasonic extraction is 60min to 100 min.

The invention also provides application of the polysaccharide SM-0.4M in preparing a product for improving immunity.

Further, the product is an immunological adjuvant.

The immune adjuvant is called adjuvant for short, namely non-specific immunoproliferative agent, and refers to auxiliary substances which can enhance the immune response capability of the body to the antigen or change the type of the immune response when being injected into the body together with the antigen or in advance.

Non-specific immunity (English: immunological immunity; internalize immunity) is also called innate immunity or innate immunity, which means that the body has a normal physiological defense function in nature, and the non-specific immune system includes: tissue barriers (skin and mucosal systems, blood-brain barrier, placental barrier, etc.); innate immune cells (phagocytes, killer cells, dendritic cells, etc.); innate immune molecules (complement, cytokines, enzymes, etc.). The composition of non-specific immunity can be summarized in three aspects: normal physiological barriers of the body; phagocytosis by phagocytic cells; normal fluid factors.

The invention also provides application of the polysaccharide SM-0.4M in preparing products for promoting macrophage proliferation or/and enhancing macrophage phagocytosis.

Macrophages (English: Macrophages, abbreviation)) Is a leukocyte located within the tissue and derived from monocytes, which in turn are derived from precursor cells in the bone marrow. Macrophages and monocytes are phagocytic cells that participate in nonspecific defense (innate immunity) and specific defense (cellular immunity) in vertebrates.

Further, the product is one or more of NO releasing agent, TNF-alpha agonist and IL-6 agonist.

The NO releasing agent refers to a product capable of promoting the release of NO from macrophages; the TNF-alpha agonist is a product capable of improving the expression of TNF-alpha and increasing the content of TNF-alpha in macrophages; the IL-6 agonist refers to a product capable of improving the expression of IL-6 and increasing the content of IL-6 in macrophages.

Further, the product is a product for preventing and/or treating tumors.

In the research on the antitumor activity of the polysaccharide, the raspberry crude polysaccharide is found to have no direct cytotoxic effect on tumor cells, but realizes the antitumor effect of the raspberry crude polysaccharide by enhancing the cellular immune reaction of tumor-bearing mice, and specifically comprises the following steps: increasing spleen index of tumor-bearing mice, and stimulating spleen cell proliferation; enhancing the activity of immune related enzymes AKP, ACP, SOD and LDH in spleen tissues of tumor-bearing mice; increasing the concentration of immune-related cytokines TNF-alpha, IL-2 and IFN-gamma in the serum of tumor-bearing mice; increase the degree of immune cell infiltration of tumor tissues, and the like.

The compound can promote the proliferation of macrophages, promote the release of NO by the macrophages, and up-regulate the expression of cell factors TNF-alpha and IL-6, further enhance the phagocytosis of the macrophages, is favorable for the macrophages to play an anti-tumor role, and can be used for preparing products for preventing and/or treating tumors.

The invention also provides application of the polysaccharide SM-0.4M in preparing a product for preventing and/or treating tumors.

The invention has the beneficial effects that:

(1) the polysaccharide component SM-0.4M is separated from raspberry polysaccharide, a specific sugar chain structure of the polysaccharide component SM-0.4M is identified, experiments verify that the polysaccharide SM-0.4M can promote macrophage proliferation, can promote the macrophage to release NO, and can up-regulate TNF-alpha and IL-6 expression, so that the phagocytosis of the macrophage is enhanced, the immunity is improved, the macrophage can play an anti-tumor role, and the raspberry polysaccharide can be used for preparing related products.

(2) The invention defines the sugar chain structures of different components in raspberry polysaccharide, and provides reference for further exploring the structure-activity relationship and developing polysaccharide antitumor drugs and immunologic adjuvants.

Drawings

FIG. 1 shows the results of the molecular weight detection of SM-0.4M;

FIG. 2 is the SM-0.4M monosaccharide composition;

FIG. 3 shows the results of SM-0.4M methylation;

FIG. 4 is an SM-0.4M infrared spectrum;

FIG. 5 is a SM-0.4M hydrogen spectrum;

FIG. 6 is a SM-0.4M carbon spectrum;

FIG. 7 is a SM-0.4M Dept135 map;

FIG. 8 is a SM-0.4M HHCOSY spectrum;

FIG. 9 is a SM-0.4M HSQC spectrum;

FIG. 10 is a SM-0.4M HMBC map;

FIG. 11 is a SM-0.4M NOESY map;

FIG. 12 is a graph showing the effect of polysaccharides on the proliferation of RAW264.7 cells;

FIG. 13 is a graph of the effect of polysaccharides on NO content of RAW264.7 cells;

FIG. 14 is a graph showing the effect of polysaccharides on TNF-. alpha.and IL-6 levels in RAW264.7 cells.

Detailed Description

The technical solutions of the present invention are described clearly and completely below, and it is obvious that the described embodiments are some, not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Extraction of raspberry crude polysaccharide

Drying the frozen raspberry fruits, crushing, removing seeds, adding 1-10 times of petroleum ether (the boiling range is 60-90 ℃) for degreasing, removing the petroleum ether, adding 85% ethanol, refluxing at 50-70 ℃ for removing monosaccharide and pigment, adding 5-30 times of purified water into the filtered filter residue, performing ultrasonic extraction (the power is 50-70W, the temperature is 50-90 ℃, the treatment time is 60-100 min), centrifuging, taking supernatant, extracting the filter residue twice according to the original condition, combining the filtrate, concentrating under reduced pressure to 1/4 of the original volume, adding 3-5 times of 95% ethanol, standing at 4 ℃ for 24h, filtering, collecting the filter residue, and performing freeze drying to obtain the crude raspberry polysaccharide.

Secondly, separating and purifying the homogeneous polysaccharide component of the raspberry

And (3) adopting DEAE Sepharose Fast Flow filler to carry out polysaccharide polarity separation and enriching neutral sugar and acidic sugar. And (3) carrying out column loading after filler pretreatment, dissolving 1g of raspberry crude polysaccharide by using distilled water, and taking a supernatant for sampling. After adjusting the flow rate, the column was eluted with distilled water, 0.2M NaCl, 0.5M NaCl, respectively, using 5 column volumes of each eluent. Performing tracing detection by phenol-sulfuric acid method, purifying, concentrating, dialyzing, freezing collected raspberry polysaccharide components, and sequentially eluting with distilled water, 0.2M NaCl and 0.5M NaCl to obtain SM-W, SM-0.2M and SM-0.4M eluents.

Molecular weight determination of raspberry polysaccharide component

Polysaccharide molecular weight and purity were determined by HPGPC. Precisely weighing a sample and a standard substance, preparing the sample into a 5mg/ml solution, centrifuging, filtering the supernatant by using a 0.22 mu m microporous filter membrane, and then transferring the supernatant into a sample injection vial. Chromatographic conditions are as follows: a chromatographic column: BRT105-104-102 series gel column (8X 300 mm); mobile phase: 0.05M NaCl solution; flow rate: 0.6ml/min, column temperature: 40 ℃; sample introduction amount: 20 mu l of the mixture; a detector: and a difference detector RI-502.

TABLE 1 SM-0.4M molecular weight test results

Fourth, determining monosaccharide composition of raspberry polysaccharide component

Monosaccharide composition was determined using an ion spectrometer (IC). 10 monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, galacturonic acid, glucuronic acid) are prepared into 10mg/ml standard solution. And (3) precisely configuring 1, 5, 10, 20, 50, 80 and 100mg/L gradient concentration standard products of the monosaccharide standard solutions to make a standard curve. 4mg of each sample was placed in an ampoule and hydrolyzed with 2M TFA at 120 ℃ for 3 h. Accurately sucking 200 μ l of acid hydrolysis solution, blowing to dry with nitrogen, adding 1ml of water, mixing uniformly by vortex, centrifuging, taking supernatant, and performing IC analysis. Chromatographic column conditions: dionex carbopaac tmp 20(3 × 150); mobile phase: a is H2O; b, 250mM NaOH; c, 50mM NaOH &500mM NaOAC; flow rate: 0.3 ml/min; sample introduction amount: 5 mu L of the solution; column temperature: 30 ℃; a detector: an electrochemical detector.

TABLE 2 SM-0.4M monosaccharide composition

Fifth, polysaccharide component ligation assay

After methylation derivatization, the polysaccharide samples were subjected to GC-MS to determine the linkage pattern. After methylation, hydrolysis and acetylation, the sample is determined by GC-MS and compared with a standard mass spectrum library.

GC-MS conditions: RXI-5SIL MS chromatography column 30 × 0.25; the temperature programming conditions are as follows: the initial temperature is 120 ℃, and the temperature is increased to 250 ℃/min at the speed of 3 ℃/min; keeping for 5 min; the temperature of the sample inlet is 250 ℃, the temperature of the detector is 250 ℃/min, the carrier gas is helium, and the flow rate is 1 mL/min.

TABLE 3 SM-0.4M methylation results

Sixthly, infrared spectrum analysis of polysaccharide components

The functional groups of the polysaccharide were analyzed by FT-IR. Precisely weighing 2mg of sample and 200mg of potassium bromide, pressing into tablets, and pressing the blank control by potassium bromide powder into tablets. Scanning and recording are respectively carried out on the FT-IR650 of the Fourier transform infrared spectrometer.

SM-0.4M Infrared results:

the absorption band is a stretching vibration absorption peak of-OH at 3600-3200cm-1, and the absorption peak in the region is a characteristic peak of the saccharide. The method comprises the following specific steps: 3426cm-1 is the O-H stretching vibration absorption peak and is a characteristic peak of saccharides. The absorption peak at 2927cm-1 is attributed to C-H stretching vibration. The peak was absorbed at 1741cm-1 and was attributed to C ═ O stretching vibration. There is an absorption peak at 1616cm-1, which is attributed to C ═ O asymmetric stretching vibration. The absorption peak is at 1421cm-1 and is attributed to C-O stretching vibration. The absorption peak is at 1103cm-1 and is attributed to C-O stretching vibration. Has an absorption peak at 1020cm-1 and is attributed to O-H stretching vibration. Has an absorption peak at 765cm-1, and may be the stretching vibration of pyran ring.

Seventhly, a polysaccharide component nuclear magnetic spectrum and a sugar chain structure

A sample of polysaccharide was weighed at 50mg, dissolved in 0.5ml of heavy water and freeze-dried. After repeating the process several times, the sample was dissolved in 0.5ml of heavy water and measured for 1H NMR spectrum, 13C NMR spectrum, DEPT135 one-dimensional spectrum and two-dimensional spectrum at room temperature and 25 ℃ in a nuclear magnetic resonance apparatus of 600MHz, as shown in FIGS. 5 to 11.

According to the above map, the sugar chain structure of SM-0.4M is as follows:

in vitro immunological activity determination of eight and three raspberry polysaccharide components

(1) Effect of Raspberry polysaccharide fraction on mouse macrophage RAW264.7 proliferation

The influence of the raspberry polysaccharide component on the proliferation of mouse macrophage RAW264.7 is detected by adopting an MTT method. RAW264.7 cells at 1X 10⁴The concentration was plated in 96-well plates. The three raspberry polysaccharide fractions were prepared to different concentrations with serum-free medium and added to well plates, with 4 multiple wells per dose. LPS (10 mug/ml) is used as a positive control, and the non-drug-added group is used as a blank control. After 24 hours of incubation, MTT assay was added. Compared with the blank control, SM-0.4M can obviously promote the proliferation of mouse macrophage RAW264.7, and the result is shown in figure 12.

(2) Effect of Raspberry polysaccharide fraction on NO content of RAW264.7 cells

RAW264.7 cells at 1X 10⁴The concentration was plated in 96-well plates. Culturing three raspberry polysaccharide fractions with serum-free mediumThe base was formulated to different concentrations and added to the well plate with 5 replicates per dose. LPS (10 mug/ml) is used as a positive control, and the non-drug-added group is used as a blank control. After 24 hours of incubation, the cell culture solution was taken and the NO content in the culture solution was measured according to the NO content measurement kit instructions. Compared with a blank control, the SM-0.4M can greatly improve the content of NO in the RAW264.7 cell culture solution, and presents obvious dose dependence, and the result is shown in figure 13.

(3) Effect of Raspberry polysaccharide component on RAW264.7 cytokine content (TNF-alpha, IL-6)

And (3) quantitatively detecting the contents of the cytokines TNF-alpha and IL-6 in the RAW264.7 cell culture solution by adopting an ELiSA kit. The specific method comprises the following steps: RAW264.7 cells at 1X 10⁴The concentration was plated in 96-well plates. The three raspberry polysaccharide fractions were prepared to different concentrations with serum-free medium and added to well plates, 5 multiple wells per dose. LPS (10 mug/ml) is used as a positive control, and the non-drug-added group is used as a blank control. After 24 hours of incubation, the cell culture medium was taken and assayed according to the kit instructions. Compared with a blank control, the SM-0.4M can greatly improve the contents of two cytokines TNF-alpha and IL-6 in the RAW264.7 cell culture solution, and the result is shown in a figure 14.

According to the test results, the polysaccharide SM-0.4M can promote macrophages to release a large amount of active factors NO, up-regulate the expression of TNF-alpha and IL-6, enhance the phagocytosis of RAW264.7 cells, facilitate the macrophages to play an anti-tumor role, and provide a basis for further research on the anti-tumor role played by raspberry polysaccharides through macrophage pathways.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.