CN107987179B - Application of a kind of low sulfated fucogalactan in the preparation of immune enhancer - Google Patents

Abstract

The invention relates to a preparation method and application of low sulfated fucan derived from marine brown algae. The low sulfated fucogalactan mainly comprises monosaccharide including galactose, fucose, glucose, etc., has weight average molecular weight of 5-30KD, and has hydrolyzed sulfate group content of 15-20%, galactose content of 40-45%, fucose content of 20-25%, and ratio of galactose content to fucose content of 2: 1. The low sulfated fucogalactan is derived from Laminaria japonica of brown algae, and has effect of activating macrophage in mouse abdominal cavity to release nitric oxide.

Description

Application of a kind of low sulfated fucogalactan in the preparation of immune enhancer

technical field

The invention relates to the preparation of biomolecules and a preparation method and application of a low sulfated fucogalactan.

Background technique

Immunity is a physiological function of the human body. The body relies on this function to identify "self" and "non-self" components, thereby destroying and rejecting pathogenic bacteria, microorganisms and other antigenic substances that enter the human body, or damaged cells and tumor cells produced by the body itself. , play a vital role in human health. The survey shows that cardiovascular disease, cancer and neurodegenerative diseases have become the three major diseases that threaten human health. Studies have shown that the immune response is likely to be used in the treatment of major diseases such as tumors, neurological diseases, cardiovascular diseases, and diabetes.

Nitric oxide (NO) plays an important role in the immune response. It belongs to the communication material between cells and is an important signal molecule for the transmission of genetic material between cells. The nitric oxide released in the body has the effect of dilating blood vessels, which can Improving cardiovascular and cerebrovascular is essential for the body to fight against bacterial invasion, remove tumor cells and inflammatory damage. Tzianabos AO reviewed the application of polysaccharides in the field of immunomodulatory activity this year, and believed that polysaccharides can effectively activate immune responses, promote the release of NO, and be further developed as immune enhancers.

The ocean is considered to be a huge potential drug storehouse, and the development and utilization of active substances from the ocean is an important part of the current understanding and utilization of the ocean. Among them, marine brown algae are rich in biomass and contain a wide variety of polysaccharides. The water-soluble polysaccharides extracted and prepared from kelp in brown algae are also complex and diverse. Different sulfated polysaccharides in kelp have been reported to have immunomodulatory effects. For example, Fang et al. reported that the water-soluble homopolysaccharide (LJP-31) isolated and prepared from kelp by column chromatography had immunomodulatory effects, and it was confirmed by structural analysis that it was mainly mannose; Sarina et al. reported a kelp Fucoidan and its fractionated components can regulate the immune function of broilers.

Our laboratory has been devoted to the development of active substances in kelp for a long time. Polysaccharides with different monosaccharide compositions have been prepared by ion exchange chromatography, one is sulfated hybrid polysaccharide, the other is fucoidan sulfate, and the other A class of sulfated galactofuccans. In a large number of activity studies, we found that the sulfate group plays an important role in it, but the higher the content, the better. It often has good activity between 10 and 30%. Secondly, the monosaccharide composition of different components affects the activity. It was found that with the increase of galactose content, the activity of polysaccharide also increased significantly. For example, among the fractions obtained by column chromatography of the low molecular weight fraction DF degraded from fucoidan sulfate, the galactose content of DF1 was significantly increased, and the activity was the best in neuron protection; kelp sulfated polysaccharide Among the fractions obtained by ion column chromatography, the F3 fraction with galactose accounting for 78.54% of the monosaccharide composition showed excellent free radical scavenging ability compared with other fractions. Therefore, we are eager to obtain a low sulfated fucogalactan whose monosaccharide composition is mainly galactose and contains a certain amount of fucose, and the preparation method should be simple and feasible, and easy to prepare in large quantities.

However, the currently prepared sulfated polysaccharides based on fucose mainly use ion chromatography classification, which is not only complicated in operation, low in yield, unable to be industrially produced, but also in the polysaccharide obtained due to the increase of eluted salt concentration. Sulfuric acid group content is high. Therefore, the present invention provides a preparation method for obtaining low-sulfated fucogalactan, and the monosaccharide composition has galactose:fucose=2:1, and shows a good immune enhancement effect; the The preparation method has simple operation and considerable yield, and can be prepared in large quantities.

SUMMARY OF THE INVENTION

In order to further explore the active ingredients in kelp, the present invention provides a preparation method of low sulfated fucogalactan, which is characterized in that the monosaccharide composition is mainly galactose, and the ratio of galactose content to fucose content About 2:1, the obtained low sulfated fucogalactan can significantly activate macrophages and promote the production of nitric oxide.

A preparation method of low sulfated fucogalactan, comprising the following steps:

Step 1, adopt the method of water extraction or acid extraction to extract water-soluble polysaccharide from kelp to make polysaccharide solution;

Step 2, adding 0.3-0.8 mol/L dilute sulfuric acid to the polysaccharide solution obtained in step 1, to a final concentration of 0.1-0.5 mol/L, and hydrolyzing at 60-100 ° C for 3-9 h under stirring;

Step 3, adding barium hydroxide to the polysaccharide solution hydrolyzed in step 2 to neutralize to neutrality, centrifuging for desalting, sedimenting the obtained degradation solution with an organic reagent, and after standing overnight, centrifuging to obtain a precipitate;

Step 4, redissolving the precipitate obtained in step 3 with water, so that the mass percentage concentration is 2-5%, to prepare an aqueous solution of the precipitate;

In step 5, after the aqueous solution of the precipitate obtained in step 4 is dialyzed with a dialysis bag, the obtained dialysate in the dialysis bag is concentrated and dried to obtain low sulfated fucogalactan;

Wherein the organic reagent is one or both of acetonitrile and acetone;

Said organic reagent sedimentation volume concentration>90%;

The molecular weight cut-off of the dialysis bag is 1KD.

Described low sulfated fucogalactan has the following characteristics:

Its main constituent monosaccharides include galactose, fucose, in addition to a small amount of glucose, mannose, rhamnose, xylose, glucuronic acid, the hydrolyzed sulfate group content is between 15-20%, and the galactose content Between 40-45%, the fucose content is between 20-25%.

The monosaccharide composition of the low sulfated fucogalactan has the characteristic that the ratio of galactose content to fucose content is about 2:1.

The molecular weight of the low sulfated fucogalactan is between 5-30KD.

The above-mentioned preparation method of the present invention has the following beneficial effects:

1 The preparation method of the present invention can obtain a low sulfated fucogalactan, and the low sulfated fucogalactan has a special monosaccharide composition, in which galactose is the main component, and fucose is the second largest. The ratio of galactose content to fucose content is about 2:1, and it also contains a small amount of sulfate groups.

2 The low sulfated fucogalactan obtained by the preparation method of the present invention can significantly activate macrophages and release nitric oxide.

Description of drawings

Fig. 1 is the preparation method schematic diagram of low sulfated fucogalactan of the present invention;

Figure 2 HPGFC molecular weight spectrum of components HDF (a) and HDFA (b) in kelp;

Figure 3 IR spectra of components HDF and HDFA in kelp;

Figure 4 Low sulfated fucogalactan promotes NO production in RAW 264.7 cells;

Figure 5. Concentration-dependent effect of hyposulfated fucogalactan HDFA on NO production in RAW 264.7 cells.

Detailed ways

The objectives, technical solutions and effects are more clear and definite, and the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention.

Embodiment 1 A kind of preparation method of low sulfated fucogalactan

Specific steps are as follows:

(1) Weigh 100g dried kelp, add 3L of water, extract at 100-120°C for 4 hours, filter with sieve and diatomaceous earth to obtain an extract, concentrate to 350mL, add final concentration 0.05mol/L magnesium chloride to remove algin , centrifuged to get the supernatant, dialysis to remove salt, concentrated and precipitated with 85% alcohol by volume, and the obtained precipitate was centrifuged and freeze-dried to obtain water-soluble polysaccharide;

(2) Weigh polysaccharide 10g and dissolve it in 200mL water, stir fully, make mass concentration of 5% polysaccharide solution; add 100mL of 0.6mol/L dilute sulfuric acid solution to it, to the final concentration of 0.2mol/L, in the case of stirring hydrolyzed at 80°C for 7h;

(3) After the degradation, add a certain amount of barium hydroxide to neutralize to pH=7, centrifuge at 4000 r/min for 15 min, discard the precipitation to obtain a degradation solution, add a certain amount of acetonitrile to the degradation solution, and make the final volume concentration of acetonitrile 90 %, after stirring overnight, the precipitate HDF was obtained after centrifugation at 4000r/min for 15min;

(4) The obtained precipitate, after freeze-drying, was reconstituted with water to make the mass percentage concentration of 2%, and the solution was put into a dialysis bag with a molecular weight cut-off of 1KD, and after dialysis with running water for 12h, hydrostatic dialysis for 4h, to obtain The dialysate in the dialysis bag is concentrated and freeze-dried to obtain low sulfated fucogalactan HDFA.

(5) chemical composition analysis is carried out to the components HDF and HDFA in the obtained kelp, including monosaccharide composition analysis, fucose content, total sugar content, sulfate group content, and molecular weight analysis, infrared analysis, the results are as follows Table 1, 2 and Figures 2 and 3.

Table 1 Chemical composition analysis of low sulfated fucogalactan

Table 2 Monosaccharide composition (molar ratio) of low sulfated fucogalactan

The HPGFC molecular weight spectrum of components HDF and HDFA (from top to bottom) in Fig. 2 kelp;

Figure 3 IR spectra of components HDF and HDFA in kelp;

It can be seen from the above results that the component HDF in kelp is a kind of complex low-sulfated heterosaccharide, and its monosaccharide composition is very complex, including galactose, fucose, glucose, rhamnose and mannose, etc. The content of various monosaccharides is abundant and no monosaccharide has a dominant position; HDFA obtained after dialysis has a significantly reduced content of heteropolysaccharides, and the monosaccharide composition is dominated by galactose, followed by fucose and glucose, and The ratio of galactose content to fucose content is 2:1, so HDFA can be regarded as a low sulfated fucogalactan.

Embodiment 2 A kind of preparation method of low sulfated fucogalactan,

(1) Weigh 100g dried kelp, add 3L of water, extract at 100-120°C for 4 hours, filter with sieve and diatomaceous earth to obtain an extract, concentrate to 350mL, add final concentration 0.05mol/L magnesium chloride to remove algin , centrifuged to get the supernatant, dialysis to remove salt, concentrated and precipitated with 85% alcohol by volume, and the obtained precipitate was centrifuged and freeze-dried to obtain water-soluble polysaccharide;

(2) take by weighing 10g of polysaccharide and dissolve it in 200mL water, stir fully, make mass concentration of 5% polysaccharide solution; add 100mL of 0.6mol/L dilute sulfuric acid solution to it, to the final concentration of 0.5mol/L, in the case of stirring hydrolyzed at 100°C for 5h;

(3) After the degradation is completed, add a certain amount of barium hydroxide to neutralize to pH=7, centrifuge at 4000 r/min for 15 min, discard the precipitation to obtain a degradation solution, add a certain amount of acetonitrile to the degradation solution, and make the final volume concentration of ethanol 90 %, stirred overnight, centrifuged at 4000 r/min for 15 min to obtain a precipitate;

(4) The obtained precipitate, after freeze-drying, was reconstituted with water to make the mass percentage concentration of 2%, and the solution was put into a dialysis bag with a molecular weight cut-off of 1KD, and after dialysis with running water for 12h, hydrostatic dialysis for 4h, to obtain The dialysate in the dialysis bag was concentrated and freeze-dried to obtain low sulfated fucogalactan (referred to as HDFB).

(5) The chemical composition of HDFB is shown in Tables 3 and 4. As can be seen from the table, the obtained component is low sulfated fucogalactan.

Table 3 Chemical composition analysis of low sulfated fucogalactan

Example 3

The difference between this embodiment and embodiment 2 is that the hydrolysis time is extended to 9 hours, and the sedimentation reagent adopts acetone, and the specific steps are:

(1) Weigh 100g dried kelp, add 3L of water, extract at 100-120°C for 4 hours, filter with sieve and diatomaceous earth to obtain an extract, concentrate to 350mL, add final concentration 0.05mol/L magnesium chloride to remove algin , centrifuged to get the supernatant, dialysis to remove salt, concentrated and precipitated with 85% alcohol by volume, and the obtained precipitate was centrifuged and freeze-dried to obtain water-soluble polysaccharide;

(2) Weigh polysaccharide 10g and dissolve it in 200mL water, stir fully, make mass concentration of 5% polysaccharide solution; add 100mL of 0.6mol/L dilute sulfuric acid solution to it, to the final concentration of 0.2mol/L, in the case of stirring hydrolyzed at 100°C for 9h;

(3) After the degradation is completed, add a certain amount of barium hydroxide to neutralize to pH=7, centrifuge at 4000 r/min for 15 min, discard the precipitation to obtain a degradation solution, add a certain amount of acetone to the degradation solution, and make the final volume concentration of acetone 90 %, stirred overnight, centrifuged at 4000 r/min for 15 min to obtain a precipitate;

(4) The obtained precipitate, after freeze-drying, was reconstituted with water to make the mass percentage concentration of 2%, and the solution was put into a dialysis bag with a molecular weight cut-off of 1KD, and after dialysis with running water for 12h, hydrostatic dialysis for 4h, to obtain The dialysate in the dialysis bag was concentrated and freeze-dried to obtain low sulfated fucogalactan (referred to as HDFC).

(5) The chemical composition of HDFC is shown in Tables 5 and 6. It can be seen from the table that the obtained component is low sulfated fucogalactan, and galactose occupies a dominant position in the monosaccharide composition.

Table 5 Chemical composition analysis of low sulfated fucogalactan

Table 6 Monosaccharide composition (molar ratio) of low sulfated fucogalactan

Example 4: Different concentrations of low sulfated fucogalactan promote NO production in RAW 264.7 cells

In this experiment, the NO release status of stimulated RAW 264.7 cells was detected by Griess reagent method.

Test samples: the low sulfated fucogalactan HDFA, HDFB, HDFC obtained in the examples.

Drawing of standard curve: Accurately prepare 1mol/L NaNO ₂ standard solution, dilute it to 80μmol/L with double distilled water, and then dilute it to the concentration of 80, 40, 20, 10, 5, 2.5, 0μmol/ L of NaNO ₂ standard solution. Pipette 100 μL of standard solution into a 96-well plate, set up three duplicate wells for each concentration, add 100 μL of Griess reagent to each well, and incubate at room temperature for 10 minutes in the dark, use a microplate reader to measure the absorbance at 550 nm, and draw a standard curve. .

Take RAW 264.7 mouse peritoneal macrophages in the logarithmic growth phase, make a cell suspension with RPMI1640 complete medium, dilute to a concentration of 1 × 105 cells/mL, and take 100 μL to inoculate a 96-well plate and place it in a 96-well plate. Incubate in a 37°C, 5% carbon dioxide incubator for 24h. After that, the medium was discarded, and 100 μL of the prepared HDFA solution was added to make the final concentration 100 μg/mL, and three replicate wells were set for each sample. The positive control group was added with LPS (lipopolysaccharide) at a final concentration of 1 μg/mL. After culturing for 24 hours, add 100 μL of equal volume of Griess reagent to each well and mix well. After incubating at room temperature for 10 minutes in the dark, use a microplate reader to measure the absorbance at 550 nm, and substitute the results into the standard curve to calculate the NO release. The results are shown in Figure 4. In the concentration gradient experiment, different concentrations of HDFA (0.01, 0.1, 1, 10 and 100 μg/mL) acted on RAW 264.7 cells for 24 h, and the Griess method was used to detect the release of NO. The results are shown in Figure 5.

The results of Griess assay showed that compared with Ctrl, LPS can effectively activate macrophages to release a large amount of NO; HDFA can also effectively activate macrophages to release a large amount of NO after co-incubating with macrophages (Fig. 4), and in a concentration-dependent manner (Fig. 5), indicating that HDFA can be used to develop immune enhancers.

Figure 4 Low sulfated fucogalactan promotes NO production in RAW 264.7 cells.

Figure 5. Concentration-dependent effect of hyposulfated fucogalactan HDFA on NO production in RAW 264.7 cells.

Claims (4)

1. Use of a low sulfated fucan in the preparation of an immunopotentiator;

the low sulfated fucan has the following characteristics: the main components of the composition are galactose, the galactose content is 40-45%, besides, the composition also comprises fucose, glucose and a small amount of mannose, rhamnose, xylose and glucuronic acid, wherein the fucose content is 20-25%, and the content of hydrolyzed sulfate groups is 15-20.5%; the low sulfated fucan has a molecular weight of 5-30 KD.

2. The use according to claim 1, wherein the low sulfated fucan is prepared by:

step 1, extracting water-soluble polysaccharide from kelp by a water extraction or acid extraction method to prepare a polysaccharide solution;

step 2, adding 0.3-0.8mol/L dilute sulfuric acid into the polysaccharide solution prepared in the step 1 until the final concentration is 0.1-0.5mol/L, and hydrolyzing for 3-9h at 60-100 ℃ under the condition of stirring;

step 3, adding barium hydroxide into the polysaccharide solution hydrolyzed in the step 2 to neutralize to be neutral, centrifuging to remove salt, settling the obtained hydrolysate with an organic reagent, standing overnight, and centrifuging to obtain a precipitate;

step 4, re-dissolving the precipitate obtained in the step 3 with water to ensure that the mass percentage concentration is 2-5% to prepare an aqueous solution of the precipitate;

step 5, dialyzing the water solution of the precipitate obtained in the step 4 by using a dialysis bag, and concentrating and drying the dialysate in the dialysis bag to obtain the low sulfated fucan;

wherein the organic reagent is one or two of acetonitrile and acetone;

the organic reagent has a sedimentation volume concentration of more than 90%;

the molecular weight cut-off of the dialysis bag is 1 KD.

3. Use according to claim 1, characterized in that: the monosaccharide composition of the low sulfated fucan has the characteristic that the ratio of the galactose content to the fucose content is about 2: 1.

4. Use according to claim 1, characterized in that: the immunopotentiator is a medicine or a health product.

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