阅读说明：本技术 硫酸化葡萄糖醛酸-木-鼠李聚糖的增强免疫功能的应用 (Application of sulfated glucuronic acid-xylo-rhamnosan in enhancing immune function ) 是由 刘英娟 郭云良 金维华 朱琳 王潇璐 王悦 武筱林 于 2019-10-11 设计创作，主要内容包括：本发明涉及生物医药领域,具体涉及一种硫酸化葡萄糖醛酸-木-鼠李聚糖的增强免疫功能的应用。该聚糖通过酸解浒苔获得,主要组成单糖包括葡糖醛酸(Glca)、木糖(Xyl)和鼠李糖(RHA)等。其制备方法：室温酸解—过滤—中和浓缩—乙醇沉淀—阴离子柱层析—水解—乙醇沉淀—Bio-Gel P-10 Gel柱层析—冷冻干燥。通过该方法制备的聚糖具有性能稳定、成本低廉、安全性高、药用价值大等特点,能够显著激活小鼠腹腔巨噬细胞释放一氧化氮。能够应用于制备免疫增强剂的药物及保健品。(The invention relates to the field of biomedicine, and in particular relates to application of sulfated glucuronic acid-xylo-rhamnosan in enhancing an immune function. The polysaccharide is obtained by acidolysis of Enteromorpha prolifera, and the main components of the polysaccharide comprise glucuronic acid (Glca), xylose (Xyl), Rhamnose (RHA) and the like. The preparation method comprises the following steps: room temperature acidolysis, filtration, neutralization and concentration, ethanol precipitation, anion column chromatography, hydrolysis, ethanol precipitation, Bio-Gel P-10Gel column chromatography and freeze drying. The glycan prepared by the method has the characteristics of stable performance, low cost, high safety, high medicinal value and the like, and can obviously activate mouse abdominal cavity macrophages to release nitric oxide. Can be used for preparing immunopotentiator medicine and health product.)

1. The application of sulfated glucuronic acid-xylo-rhamnosan in enhancing immune function is characterized in that the specific operation steps are carried out as follows:

s1, extracting enteromorpha crude polysaccharide: weighing 1000g of dry Enteromorpha prolifera, adding 30L of 0.1M HCl, extracting at room temperature for 4h to obtain an extracting solution, filtering the extracting solution with diatomite, centrifuging after neutralization to obtain a supernatant, concentrating the supernatant, precipitating with ethanol, and determining that the yield of crude polysaccharide (EP) is 20.1%;

s2, DEAE-Bio Gel Agarose FF ion column chromatography of crude polysaccharide: subjecting 8g of crude polysaccharide (EP) to DEAE-Bio GelAgarose FF column chromatography for fractionation, using NaCl with different concentrations as eluent, collecting fractions to obtain a water-washing fraction (EP1), a 0.3M NaCl fraction (EP2), a 1M NaCl fraction (EP3) and a 2M NaCl fraction (EP4), collecting each fraction, dialyzing for desalination, and concentrating and precipitating with ethanol to obtain each fraction;

s3, preparation of sulfated glucuronic acid-xylo-rhamnosan: hydrolyzing 1M NaCl fraction (EP3), separating by Bio-GelP-10Gel chromatography (2.6X 100cm), and separating 0.5M NH₄HCO₃As eluent, 2 fractions were obtained, namely: dialyzing and desalting the high molecular weight component (DEP1) and the low molecular weight component (DEP2), concentrating under reduced pressure, and lyophilizing at low temperature to obtain sulfated glucuronic acid-xylo-rhamnosan (DEP 2).

2. The use of sulfated glucuronic acid-xylan-rhamnosan for enhancing immune function, as claimed in claim 1, wherein the cut-off molecular weight of the dialysis bag used for dialysis desalting is 1 kD.

3. The use of sulfated glucuronic acid-xylan-rhamnosan for enhancing immune function, as claimed in claim 1, wherein the molecular weight of sulfated glucuronic acid-xylan-rhamnosan is 17.3 kD.

4. The use of sulfated glucuronic acid-xylan-rhamnosan for enhancing immune function according to any one of claims 1 to 3, wherein the sulfated glucuronic acid-xylan-rhamnosan is effective in activating mouse peritoneal macrophages to release nitric oxide, activating inflammasome NLRP3 pathway, and enhancing immunoregulatory ability of macrophages.

5. The application of the sulfated glucuronic acid-xylo-rhamnosan for enhancing the immune function according to claim 4, wherein the prepared sulfated glucuronic acid-xylo-rhamnosan can be used as an active ingredient for preparing drugs and health products of immunopotentiators.

The technical field is as follows:

the invention belongs to the technical field of biological medicines, and particularly relates to application of sulfated glucuronic acid-wood-rhamnosan derived from sea enteromorpha in enhancing an immune function.

Background art:

the immunity is an extremely complex physiological reaction, and is a function of maintaining the physiological balance and stability of the organism by recognizing and eliminating heterogeneous molecules by the immune system of the organism. The body relies on this function to recognize "self" and "non-self" components to combat tissue damage and infection caused by pathological agents such as pathogens, toxic compounds or radiation. This process involves the involvement of inflammatory mediators, inflammation-related signaling pathways, and inflammatory cytokines.

Abundant biological resources are stored in the ocean, and the special growth and reproduction modes and adaptation mechanisms of marine organisms are caused by the high-salt, high-pressure, anoxic, low (constant) temperature, low (no) illumination and oligotrophic environment of the marine water body, wherein a large number of natural active substances with complexity, variety, novel structure and unique activity exist. Marine algae, as an important source of unique bioactive substances, can be used as a new source of medicaments and has great significance in the medical field. Enteromorpha (Enteromorpha prolifera) is an aquatic algae plant in green algae ulva, is used as a natural seaweed in sea areas, and has extremely high yield. Enteromorpha polysaccharide is a key active ingredient of Enteromorpha and has been proved to have various biological activities, such as oxidation resistance, antibiosis, hyperlipemia resistance, glucose metabolism regulation and the like. Kim et al found that polysaccharides obtained by hot water extraction had an immunomodulatory effect, but the mechanism of action was not clear. Therefore, the invention seeks to design and provide the application of the sulfated glucuronic acid-wood-rhamnosan for enhancing the immune function, the sulfated glucuronic acid-wood-rhamnosan oligosaccharide with the molecular weight of 17.3kD is further degraded and separated by the enteromorpha polysaccharide obtained by acidolysis, the structural composition of the oligosaccharide is inconsistent with the structure of the enteromorpha polysaccharide reported in the past, and the research on the immune regulation function activity of the oligosaccharide is not reported yet.

The invention content is as follows:

the object of the present invention is to overcome the above mentioned drawbacks of the prior art and to seek to provide an application of sulfated glucuronic acid-xylo-rhamnosan for enhancing immune function.

In order to achieve the purpose, the invention relates to an application of sulfated glucuronic acid-xylo-rhamnosan in enhancing immune function, which is realized by the following technical scheme:

s1, extracting enteromorpha crude polysaccharide: weighing 1000g of dry Enteromorpha prolifera, adding 30L of 0.1M HCl, extracting at room temperature for 4h to obtain an extracting solution, filtering the extracting solution with diatomite, centrifuging after neutralization to obtain a supernatant, concentrating the supernatant, precipitating with ethanol, and determining that the yield of crude polysaccharide (EP) is 20.1%;

s2, DEAE-Bio Gel Agarose FF ion column chromatography of crude polysaccharide: taking 8g of crude polysaccharide (EP) to carry out DEAE-BioGel Agarose FF column chromatography fractionation, taking NaCl with different concentrations as eluent, collecting components to obtain a water washing component (EP1), a 0.3M NaCl component (EP2), a 1M NaCl component (EP3) and a 2M NaCl component (EP4), collecting each component, dialyzing and desalting, and concentrating and precipitating with ethanol to obtain each fractionated component;

s3, preparation of sulfated glucuronic acid-xylo-rhamnosan: hydrolyzing 1M NaCl fraction (EP3), separating by Bio-Gel P-10Gel chromatography (2.6X 100cm), and separating by 0.5M NH₄HCO₃As eluent, 2 fractions were obtained, namely: dialyzing and desalting the high molecular weight component (DEP1) and the low molecular weight component (DEP2), concentrating under reduced pressure, and lyophilizing at low temperature to obtain sulfated glucuronic acid-xylo-rhamnosan (DEP 2).

Further, the cut-off molecular weight of the dialysis bag used for dialysis desalination is 1 kD;

further, the sulfated glucuronic acid-xylo-rhamnosan of the present invention has a molecular weight of 17.3 kD.

Furthermore, the sulfated glucuronic acid-wood-rhamnosan can effectively activate mouse abdominal cavity macrophages to release nitric oxide, activate inflammatory corpuscle NLRP3 channels and enhance the immunoregulation capability of the macrophages.

Furthermore, the sulfated glucuronic acid-wood-rhamnosan prepared by the invention can be used as an active ingredient to be applied to preparation of immunopotentiators and health products.

Compared with the prior art, the invention has the following beneficial effects: the enteromorpha prolifera is used as a preparation raw material, the raw material is environment-friendly and easy to obtain, and meanwhile, the preparation process has simple steps and high preparation efficiency. Meanwhile, the prepared target product can effectively activate an inflammatory corpuscle NLRP3 pathway.

Description of the drawings:

FIG. 1 is a schematic diagram of the principle of the preparation method of sulfated glucuronic acid-xylo-rhamnose oligosaccharide related to the invention.

FIG. 2 is a schematic diagram of the release of NO from RAW264.7 cells promoted by sulfated glucuronic acid-xylo-rhamnose oligosaccharide.

FIG. 3 is a schematic diagram showing the concentration dependence of sulfated glucuronic acid-xylo-rhamnose oligosaccharide on the NO release amount of RAW264.7 cells.

FIG. 4 is a schematic diagram of the activation of small bodies of RAW264.7 cells NLRP3 by sulfated glucuronic acid-xylo-rhamnose oligosaccharide related to the invention.

The specific implementation mode is as follows:

the following detailed description is made by way of example with reference to the accompanying drawings. Those skilled in the art will appreciate that these embodiments are illustrative, not restrictive. These examples are not intended to limit the scope of the present invention in any way.