阅读说明：本技术 一种免疫调节剂的制备方法 (Preparation method of immunomodulator ) 是由 刘金玲 肖美添 叶静 张娜 李航 于 2021-10-20 设计创作，主要内容包括：本发明公开了一种免疫调节剂的制备方法,采用响应面(RSD)法优化提取工艺,从海带中提取岩藻聚糖,并通过乙醇重沉淀法和Q sepharose fast flow(Q FF)阴离子交换柱层析分离纯化岩藻聚糖,最终得到四个纯度较高的岩藻聚糖组分：SF1、SF2、SF3、SF4。并且评价了岩藻聚糖SF1、SF2、SF3、SF4对RAW 264.7细胞毒性、NO释放、促炎细胞因子分泌以及MAPK信号通路中ERK 1/2、JNK和p38蛋白表达水平的影响。结果表明：SF1显著促进NO释放,抑制促炎细胞因子分泌,上调ERK 1/2、JNK和p38磷酸化水平。以上结果说明SF1有望成为免疫疾病或功能性食品的新型免疫调节剂。(The invention discloses a preparation method of an immunomodulator, which adopts a Response Surface (RSD) method to optimize an extraction process, extracts fucosan from kelp, separates and purifies the fucosan through an ethanol reprecipitation method and Q sepharose fast flow (Q FF) anion exchange column chromatography, and finally obtains four fucosan components with higher purity: SF1, SF2, SF3, SF 4. And the effect of fucoidan SF1, SF2, SF3, SF4 on RAW264.7 cytotoxicity, NO release, pro-inflammatory cytokine secretion and expression levels of ERK 1/2, JNK and p38 proteins in the MAPK signaling pathway were evaluated. The results show that: SF1 significantly promotes NO release, inhibits proinflammatory cytokine secretion, and up-regulates the phosphorylation levels of ERK 1/2, JNK and p 38. The above results indicate that SF1 is expected to be a novel immunomodulator for immune diseases or functional foods.)

1. A method for preparing an immunomodulator, which is characterized by comprising the following steps:

step 1, extracting a fucosan crude product from kelp by adopting a Response Surface (RSD) optimized water extraction method to prepare a polysaccharide solution;

step 2, adding ethanol into the polysaccharide solution prepared in the step 1 until the volume fraction is 30%, and centrifuging (10000r/min, 20min) until the supernatant is clear and transparent;

step 3, adding ethanol into the supernatant obtained in the step 2 until the volume fraction is 70%, centrifuging to obtain a precipitate, and washing the precipitate for 3 times by using absolute ethyl alcohol;

step 4, freeze-drying the precipitate washed in the step 3 to obtain a crude fucosan (SF);

and 5, separating and purifying fucosan from the SF obtained in the step 4 through QFF column chromatography, measuring the absorbance of each tube by adopting a phenol-sulfuric acid method, and drawing an elution curve according to the tube number to the absorbance. Respectively collecting fucosan components according to the elution peak, dialyzing until no NaCl exists, concentrating, and freeze-drying.

2. The immunomodulator according to claim 1, wherein the main monosaccharide composition is fucose, and purification results in four fucosan components of higher purity: SF1, SF2, SF3, SF4, fucose contents of 43.96%, 40.02%, 38.62% and 27.83%, sulfate contents of 12.05%, 15%, 23.79% and 36.94%.

3. The immunomodulator according to claim 1, wherein said high purity fucoidan is analyzed by HPLC for molecular weights of four components, the average molecular weights being 225.28kDa, 231.72kDa, 234.68kDa and 249.68kDa, respectively.

4. The method of claim 1, wherein the fucoidan fraction SF1 is substantially comprised of D- (+) -mannose, L- (+) -rhamnose, D- (+) -galactose, and L- (-) -fucose in a molar ratio of 0.6249: 0.0556: 0.8408: 1.

5. The immunomodulator according to claim 1, wherein said immunomodulator substantially promotes the secretion of NO in RAW 264.7.

Technical Field

The invention relates to the technical field of medicine preparation, in particular to a preparation method of an immunomodulator.

Background

The immune system (innate immunity and adaptive immunity) has immune surveillance, defense and regulation effects. Innate immunity is the first line of defense and does not require the first encounter with pathogens or other foreign substances to immediately respond to an intruder. Besides the skin and mucosal barriers, phagocytic cells such as monocytes, macrophages and neutrophils are also innate immunity. The immunity is a physiological function of human body, and the organism can distinguish self and non-self components by means of the function, so that antigen substances such as pathogenic bacteria and microorganisms entering the human body or damaged cells and tumor cells generated by the organism can be destroyed and rejected, and the immunity plays a vital role in human health. Investigations have shown that cardiovascular diseases, cancer and neurodegenerative diseases are three major diseases threatening human health. The existing research shows that the immune response is possibly applied to the treatment of serious diseases such as tumor, nervous system diseases, cardiovascular diseases, diabetes and the like. In reports investigating polysaccharide immunostimulatory activity, most studies have involved macrophages. Immunomodulatory polysaccharides can increase the production of Reactive Oxygen Species (ROS), Nitric Oxide (NO), and the secretion of proinflammatory cytokines such as TNF- α, Interleukin (IL) -1, IL-6, IL-8, IL-12, and the like.

The ocean is considered as a huge potential drug reservoir, and the development and utilization of active substances from the ocean is an important content for understanding and utilizing the ocean at present. The marine brown algae is rich in biomass and contains various polysaccharides, and the water-soluble polysaccharides extracted and prepared by taking kelp in the brown algae as a research object are also complex and various. Research has reported that different sulfated polysaccharides in kelp have immune regulation effects. For example, Qiao et al found that fucoidan has a higher content of sulfate groups and is associated with a higher immunostimulatory activity of macrophages. In addition, the study by Khilchenko et al found that almost all of the activity of the prepared fucoidan was lost by deacetylation and desulfation, indicating that the presence of both acetyl and sulfate groups is critical to the biological activity of fucoidan.

Disclosure of Invention

1. Technical problem to be solved

The invention aims to provide a convenient and rapid method for extracting and separating fucosan with immunoregulatory activity from kelp.

2. Technical scheme

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for preparing an immunomodulator, comprising the following steps:

step 1, extracting a fucosan crude product from kelp by a water extraction method to prepare a polysaccharide solution;

step 2, adding ethanol into the polysaccharide solution prepared in the step 1 until the volume fraction is 30%, and centrifuging (10000r/min, 20min) until the supernatant is clear and transparent;

step 3, adding ethanol into the supernatant obtained in the step 2 until the volume fraction is 70%, centrifuging to obtain a precipitate, and washing the precipitate for 3 times by using absolute ethyl alcohol;

step 4, freeze-drying the precipitate washed in the step 3 to obtain a crude fucosan (SF);

and 5, separating and purifying fucosan from the SF obtained in the step 4 through QFF column chromatography, measuring the absorbance of each tube by adopting a phenol-sulfuric acid method, and drawing an elution curve according to the tube number to the absorbance. Respectively collecting fucosan components according to the elution peak, dialyzing until no NaCl exists, concentrating, and freeze-drying.

Preferably, the main component monosaccharide is fucose, and four fucosan components with higher purity are finally obtained by purification: SF1, SF2, SF3 and SF4, containing 43.96%, 40.02%, 38.62% and 27.83% fucose and 12.05%, 15%, 23.79% and 36.94% sulfate radical, respectively.

Preferably, the high purity fucoidan is analyzed by HPLC for molecular weight of each component, and the average molecular weight is 225.28kDa, 231.72kDa, 234.68kDa and 249.68kDa respectively.

Preferably, the fucosan component SF1 mainly comprises D- (+) -mannose, L- (+) -rhamnose, D- (+) -galactose and L- (-) -fucose, and the molar ratio is 0.6249: 0.0556: 0.8408: 1.

Preferably, the high-purity fucosan SF1 can remarkably promote the secretion of NO and cytokines in RAW264.7, and can remarkably up-regulate the expression levels of ERK 1/2, JNK and p38 phosphorylation proteins.

3. Advantageous effects

Compared with the prior art, the invention has the advantages that:

in the invention, the prepared fucosan component SF1 can remarkably promote the secretion of NO and cytokines in RAW264.7, and simultaneously, the expression levels of ERK 1/2, JNK and p38 phosphorylation proteins are remarkably increased. The above results indicate that SF1 is expected to be a novel immunomodulator for immune diseases or functional foods.

Drawings

Figure 1 is an HPLC diagram of PMP derivatives of acid hydrolysates of different fucosan fractions.

FIG. 2 is an infrared spectrum of a fucosan component of kelp.

FIG. 3 is a UV spectrum of fucosan from Laminaria japonica.

Figure 4 is a graph of the effect of fucoidan on RAW264.7 cytotoxicity.

FIG. 5 is the effect of fucoidan on LPS-induced NO release from RAW264.7 cells.

FIG. 6 is a graph showing the effect of different fucoidans on the secretion of the cytokine TNF-. alpha..

FIG. 7 shows the effect of different fucoidans on the secretion of the cytokine IL-1 β.

FIG. 8 is a graph of the effect of different fucoidans on cytokine IL-6 secretion.

FIG. 9 is a graph of the effect of SF1 on the levels of ERK 1/2, JNK and p38 proteins in RAW264.7 cells;

FIG. 10 is a graph of the effect of SF4 on ERK 1/2, JNK and p38 protein levels in RAW264.7 cells.

Detailed Description

The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments.

Example 1:

a method for preparing an immunomodulator, comprising the following steps:

step 1: weighing 5g of kelp powder which flows back by ethanol, adding a certain volume of distilled water, and extracting for a certain time at a certain temperature. After extraction, the extract is filtered through a 200-mesh filter cloth while the extract is hot to remove precipitates, 1/4 volumes of 4% calcium chloride solution are added into the supernatant to remove alginic acid in the kelp extract, and centrifugation is carried out after impurities are completely precipitated (4000r/min, 15 min). Concentrating the supernatant after removing impurities, and adding 95% ethanol to make the volume fraction of ethanol in the solution 70%. And (3) after the precipitation is complete, centrifuging (10000r/min, 5min), precipitating, washing the precipitate for 3 times by using absolute ethyl alcohol, freezing and drying to obtain a fucosan crude product, and calculating the extraction rate of the fucosan crude product.

Step 2: a Response Surface (RSD) method is adopted to optimize a fucosan extraction process, and the influence of extraction temperature (60, 70, 80, 90 and 100 ℃), extraction time (1, 2, 3, 4, 5 and h) and liquid-material ratio (20: 1, 30: 1, 40: 1, 50: 1 and 60: 1) on the fucosan rate is examined. Through optimization, the optimal extraction conditions of water extraction are as follows: the extraction temperature is 90 ℃, the extraction time is 4 hours, and the liquid-material ratio is 40: 1; under the optimal condition, three times of repeated experiments are respectively carried out to obtain the fucosan with the extraction rate of 9.38%;

and step 3: and (3) purifying fucosan:

3.1: ethanol reprecipitation method: dissolving appropriate amount of fucosan crude product in distilled water, adding ethanol until volume fraction is 30%, and repeating centrifugation (10000r/min, 20min) step until supernatant is clear and transparent. Collecting supernatant, adding ethanol until volume fraction is 70%, centrifuging to obtain precipitate, washing the precipitate with anhydrous ethanol for 3 times, and freeze drying the washed precipitate to obtain crude fucosan (SF).

3.2: separating and purifying fucosan sample by Q sepharose fast flow anion exchange column chromatography, namely preparing the fucosan sample into a solution with the concentration of 100mg/mL and filtering the solution through a 0.45 mu m filter membrane. In the experiment, fucosan is separated and purified by Q FF column chromatography, 100mL of Q FF is taken and filled into a column, 5 column volumes are balanced by distilled water, 0.3mL of sample solution is added, Na Cl solutions with different concentrations are respectively prepared for isocratic elution of Q FF, the flow rate is 2mL/min, and each tube is 4 mL. And (3) measuring the absorbance of each tube by adopting a phenol-sulfuric acid method, and drawing an elution curve by using the number of the tubes to the absorbance. Respectively collecting fucosan components according to the elution peak, dialyzing, concentrating and freeze-drying.

And further purifying SF by adopting an ethanol re-precipitation method and Q FF anion exchange column chromatography to finally obtain four fucosan components with higher purity. The obtained fractions were subjected to chemical composition analysis including monosaccharide composition analysis, fucose content, total sugar content, sulfate group content, molecular weight analysis, etc., and the results are shown in tables 1 and 2 below.

Table 1 physicochemical properties of crude fucosan SF:

table 2 physicochemical properties of the fucoidan components SF 1-SF 4:

in the invention, the average molecular weight of the fucosan component is determined by HPLC, and the average molecular weights of the components SF 1-SF 4 are 225.28kDa, 231.72kDa, 234.68kDa and 249.68kDa respectively.

In the invention, FIG. 1 is an HPLC diagram of PMP derivatives of acid hydrolysis products of different fucosan components, wherein the molar ratio of monosaccharides of the fucosan component is SF1 which mainly comprises D- (+) -mannose, L- (+) -rhamnose, D- (+) -galactose and L- (-) -fucose, and the molar ratio is 0.6249: 0.0556: 0.8408: 1; SF2 is mainly composed of D- (+) -mannose, L- (+) -rhamnose, D- (+) -galactose, D- (+) -xylose and L- (-) -fucose, and the molar ratio is 0.4962: 0.8136: 0.4747: 0.0460: 1; SF3 is mainly composed of D- (+) -mannose, L- (+) -rhamnose, D- (+) -galactose, D- (+) -xylose and L- (-) -fucose, and the molar ratio is 0.2782: 0.6289: 0.5761: 0.0553: 1; SF4 is composed of D- (+) -mannose, L- (+) -rhamnose, D- (+) -galactose, L- (-) -fucose, in a molar ratio of 0.7369: 0.6787: 1.7292: 1 (less than 1% molar is considered as trace).

In the invention, fig. 2 is an infrared spectrogram of a kelp fucosan component, and the infrared spectrogram shows that: fucoidan SF and SF 1-SF 4 are sulfated polysaccharides, the substitution position of the sulfuric acid group is mainly the axial C4 position, and the polysaccharides contain acetyl groups.

In the present invention, fig. 3 shows by ultraviolet spectrum: SF 1-SF 4 do not contain proteins or nucleic acids.

Example 2:

the effect of fucosan SF1 on RAW264.7 cytotoxicity, NO release, pro-inflammatory cytokine secretion, and expression levels of ERK 1/2, JNK, and p38 proteins in the MAPK signaling pathway.

1. Cytotoxicity assays

SF 1-SF 4 were diluted to different concentrations with growth medium. RAW264.7 cells were cultured in growth medium at 37 ℃ and then seeded into 96-well plates (1X 10)⁴Cells/well) and treated by adding increasing concentrations of SF1(50-200 μ g/mL) to 96-well plates, respectively.

After 24h incubation, cell viability was determined using the MTT method: the cell supernatant was first removed and 100. mu.L of MTT solution (0.5mg/mL) was added to each well and incubated for an additional 4 h. Next, the supernatant was discarded and 100 μ L DMSO was added to the cell culture, followed by shaking in the dark for 10min until no visible particulate matter was detected. The absorbance at 570nm was measured using a microplate reader. The results of the toxic effect of SF1 on macrophages are shown in FIG. 4. SF1 has no toxic effect on macrophages and has promoting effect within the concentration range of 50-200 mug/mL.

2. NO production amount measurement (Griess method)

SF 1-SF 4 were diluted to different concentrations with growth medium. RAW264.7 cells were plated in 48-well plates (1X 10)⁵Individual cells/well) were preincubated at 37 ℃ for 24 h. Increasing concentrations of SF1 (50-200. mu.g/ml) were then added to the well plates for 24h treatment. Meanwhile, cells treated with LPS (1. mu.g/mL) and dexamethasone (DEM, 10ng/mL) under the same conditions were used as a control group. The cell culture supernatant was then collected and mixed with an equal volume of Griess reagent, incubated at room temperature for 30min, and measured with a microplate reader at 540 nm. The results are shown in FIG. 5.

As can be seen from fig. 5, NO production in the blank is much lower than in LPS and DEM treated groups with significant variability. Meanwhile, after different concentrations of SF1 are treated for 24 hours, the generation of NO is concentration-dependent, and the NO release amount of macrophages is gradually increased. The experimental result shows that SF1 has a remarkable stimulation effect on RAW264.7 cells, and particularly has a promoting effect on the generation of NO.

3. Proinflammatory cytokine assay

SF 1-SF 4 were diluted to different concentrations with growth medium. RAW264.7 cells were plated in 6-well plates (1X 10)⁵Individual cells/well) were preincubated at 37 ℃ for 24 h. Increasing concentrations of SF1 (50-200. mu.g/ml) were then added to the well plates for 24h treatment. Meanwhile, cells treated with LPS (1. mu.g/mL) under the same conditions were used as a control group. Then, cell culture supernatant was collected and the contents of TNF-. alpha.IL-1. beta., IL-6 were determined using the kit. The results are shown in FIGS. 6-8.

As can be seen from FIGS. 6-8, TNF-. alpha.IL-1. beta., IL-6 production in the blank was much lower than in the LPS-treated group with significant variability. Meanwhile, after the treatment of SF1 with different concentrations for 24h, the release amount of TNF-alpha, IL-1 beta and IL-6 of the macrophage is gradually increased. The experimental result shows that SF1 can promote TNF-alpha, IL-1 beta and IL-6 secretion of RAW264.7 cells.

Determination of the expression levels of ERK 1/2, JNK and p38 proteins in the MAPK signaling pathway

SF 1-SF 4 were diluted to different concentrations with growth medium. RAW264.7 cells were plated in 6-well plates (1X 10)⁵Individual cells/well) were preincubated at 37 ℃ for 24 h. Increasing concentrations of SF1 (50-200. mu.g/ml) were then added to the well plates for 24h treatment. At the same time, LPS (1. mu.g/mL) was used for treatment under the same conditionsThe cells of (3) were used as a control group. Then, the cells were collected, cell lysates were prepared, and concentrations of ERK 1/2, JNK and p38 and phosphorylated ERK 1/2, JNK and p38 proteins were determined therein. The results are shown in FIG. 9.

As can be seen from fig. 9, LPS (1 μ g/mL) significantly promoted the levels of ERK 1/2, JNK, p38 phosphorylation in RAW264.7 cells, however, expression of non-phosphorylated ERK 1/2, JNK, p38 protein levels was not affected by LPS or the combination of LPS and SF 1. With increasing concentrations of SF1, p-ERK 1/2, p-JNK, and p-p38 all showed dose-dependent increases, suggesting that SF1 may modulate immune responses by stimulating MAPK pathways in macrophages. The above results indicate that SF1 is expected to be a novel immunomodulator for immune diseases or functional foods.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and equivalent alternatives or modifications according to the technical solution and the inventive concept of the present invention should be covered by the scope of the present invention.