WO2017008768A1 - Glucan and preparation method thereof, and application in preparation of immune-enhancing and antitumor medicine and functional food - Google Patents

Abstract

Provided are a glucan and preparation method thereof, and an application in the preparation of immune-enhancing and antitumor medicine and functional food. The method comprises: drying and crushing Antarctic brown algae; and after degreasing, water extraction, precipitation by calcium chloride, and purification by an anion exchange resin, obtaining a glucan having β-1,3-glucose as a main chain, β-1,6-glucose as a branched chain, and a molecular weight of 1.1 kDa-50 kDa. The β-glucan prepared in the present invention has immune-enhancing activities indicated by in vitro neutral red uptake of macrophages, and T and B lymphocyte proliferation tests, and also has functions of tumor growth suppression, tumor metastasis inhibition, and an increase in a spleen index of tumor-bearing mice immune system organs indicated by experiments.

Description

Glucan and preparation method thereof and application thereof in preparing medicines and functional foods for immune enhancement and anti-tumor Technical field

The invention belongs to the field of marine medicine, and relates to a method for preparing high-purity low-molecular-weight glucan by using seaweed and application thereof, in particular to a glucan and a preparation method thereof, and a medicament and a function for preparing for immune enhancement and anti-tumor. Food applications.

Background technique

Glucan is a polysaccharide material with glucose as the sole component. It is widely found in animals, plants and microorganisms as well as brown algae. There are many kinds of glucans in nature, and there are linear or branched α/β-glucans. The connection between glucose is 1,2-linkage, 1,3-linkage, 1,4-linkage and 1, 6-linked form, in which β-1,3 and β-1,6-glucan have good biological activities, such as anti-tumor, enhance immunity, promote wound healing, anti-radiation and prevent hyperlipidemia. According to the World Health Organization, in the next 20 years, cancer cases will rise from 14 million in 2012 to 22 million. In China, 8,550 people become cancer patients every day, and one out of every seven to eight people die of cancer. Cancer Treatment In addition to surgery and radiotherapy, immunotherapy is one of the safe and reliable therapies, and the market demand for anti-tumor β-1,3-glucan by immunopotentiation is large. In addition to its use as an immunopotentiating and anti-tumor effect, β-1,3-glucan has a wide range of uses in the fields of substitute plasma, prevention of oral mucosal ulcers and cosmetics, and has broad market applications.

At present, β-1,3-glucan on the market mostly comes from terrestrial organisms such as barley, oats, edible fungi (chanterelle, ash tree, schizophyllum), yeast, etc., due to different sources of raw materials, the resulting β-1, The molecular weight, the connection mode and the branching degree of 3-glucan are very different, and the quality is difficult to control. For example, the β-glucan for injection medicine is mainly derived from shiitake mushroom, and is a β-1,6-branched group. --1,3-glucan has poor water solubility due to its molecular weight of up to 400-800 kDa. With the continuous expansion of the application field of β-glucan and the increasing market demand, the existing mushroom β-glucan can no longer meet the needs of the market.

In the preparation of seaweed-derived glucan, a Chinese invention patent (Patent No. 201010169739.7) was found to involve the preparation method of seaweed low molecular weight glucan. The raw materials are kelp, wakame and sarcophagus. It is a method of fractional precipitation of 80% ethanol and oxidation of hydrogen peroxide. The obtained product has a molecular weight of only 800-1000 Daltons (Da), the structure is not clear, and no activity report inhibiting tumor growth and metastasis is reported.

Summary of the invention

It is an object of the present invention to provide a glucan, a process for the preparation thereof and use thereof for the preparation of a medicament for immunopotentiating and antitumor, said β-1,3/1,6-glucan Sugar is prepared from Antarctic brown algae. Both cell and animal experiments show that it is safe, non-toxic, and has significant immune enhancement and anti-tumor activity. It has the potential to be developed for immune enhancement and anti-tumor effects, and can make up for market medicinal beta. The need for -1,3/1,6-glucan deficiency.

In order to achieve the above object, the technical solution of the present invention is:

The present invention provides a glucan which is β-1,3/1,6-glucan, which has β-1,3-glucose as the main chain and β-1,6- Glucose is branched, and the weight average molecular weight is 1.1 kDa to 50 kDa; the content of glucose in the β-1,3/1,6-glucan is 90 wt% to 98 wt%, and the crude protein content is 0.5% wt-2.5% wt. The rest is moisture.

The β-1,3/1,6-glucan is extracted from Antarctic brown algae, which is sea velvet, sea bamboo shoot or rosenbergea.

The invention provides a preparation method of the glucan, which comprises the following steps:

(1) Degreasing: drying and pulverizing Antarctic brown algae to obtain algal flour, soaking and stirring with an organic solvent to obtain defatted algal flour;

(2) Water extraction: the degreased algae powder is extracted with water at room temperature to obtain an aqueous extract;

(3) Classification: The aqueous extract obtained in the step (2) is centrifuged, and an aqueous calcium chloride solution is added to the supernatant obtained by centrifugation; after stirring, the mixture is centrifuged, and the supernatant is taken for dialysis or ultrafiltration desalting, and concentrated under reduced pressure to obtain Crude polysaccharide

(4) Purification: the crude polysaccharide of step (3) is dissolved in distilled water, and distilled water and sodium chloride aqueous solution are used as The mobile phase was separated and purified by anion exchange resin, detected by a phenolic acid method, and the water-eluting fraction was collected, concentrated under reduced pressure, and lyophilized to obtain the β-1,3/1,6-glucan.

Further, the ratio of the volume of the organic solvent added in the step (1) to the mass ratio of the algal powder is 10 to 20:1, and degreasing by immersion or intermittent stirring for 2 to 8 hours, and the organic solvent used is ethanol, acetone or methanol. One or several.

Further, the volume of the calcium chloride aqueous solution added in the step (3) is 1 to 3 times the volume of the supernatant, the concentration of the calcium chloride aqueous solution is 1-3 mol/L, and the membrane for the dialysis or ultrafiltration is retained. The molecular weight is from 1,000 to 3,000 Da.

Further, the concentration of the aqueous solution of sodium chloride used in the purification of the anion exchange resin in the step (4) is 0.01 to 2.0 mol/L, and elution is 2 to 8 column volumes.

The present invention also provides the use of the β-1,3/1,6-glucan for the preparation of a medicament for immunoenhancement and antitumor and a functional food.

Further, when the concentration of the β-1,3/1,6-glucan is from 1 to 100 μg/mL, the macrophage is promoted to phagocytose neutral red.

Further, when the β-1,3/1,6-glucan has a concentration of 1 to 200 μg/mL, it has an effect of promoting proliferation of T and B lymphocytes.

Further, when the β-1,3/1,6-glucan dose is greater than 0.5 mg/kg/day, the tumor growth and tumor metastasis of the tumor-bearing mice can be effectively inhibited.

Further, when the β-1,3/1,6-glucan dose is greater than 0.5 mg/kg/day, the index of the immune organs of the tumor-bearing mice can be effectively increased.

Further, the β-1,3/1,6-glucan is compounded with paclitaxel, cisplatin or fluorouracil, and docetaxel for preparing an antitumor drug.

The advantages and benefits of the present invention are:

(1) The present invention dries and pulverizes Antarctic seaweed, and is purified by degreasing, calcium chloride precipitation and anion exchange resin to obtain β-1,3-glucose as a main chain and β-1,6-glucose in high purity. Branched low molecular weight -1-1,3/1,6-glucan having a glucose content of 90% by weight to 98% by weight, a protein content of 0.5% by weight to 2.5% by weight, and a weight average molecular weight of 1.1 kDa to 50 kDa. The raw material is derived from edible brown algae in Antarctica and has high safety.

(2) The raw material resources used in the invention are abundant, the preparation process is simple, the cost is low, and the industrial production is easy, which can make up for the defects of insufficient market supply.

(3) The β-1,3/1,6-glucan prepared by the method of the invention has a purity of 90 wt% to 98 wt% and a molecular weight of 1.1 kDa to 50 kDa.

(4) The low molecular weight β-1,3/1,6-glucan prepared by the method of the present invention has immunopotentiation and inhibits tumor growth and metastasis at the cell and animal levels.

DRAWINGS

Fig. 1 is a HPLC diagram showing the monosaccharide composition analysis of β-1,3/1,6-glucan of the present invention.

2 is a Fourier transform infrared spectroscopy (FTIR) image of the β-1,3/1,6-glucan of the present invention.

Fig. 3 is a nuclear magnetic resonance carbon spectrum ( ¹³ C-NMR) chart of β-1,3/1,6-glucan of the present invention.

Figure 4 is a graph showing the effect of β-1,3/1,6-glucan on promoting phagocytosis of neutral red by macrophages of the present invention.

Fig. 5 is a graph showing the effect of β-1,3/1,6-glucan on the proliferation of B lymphocytes (5A) and T lymphocytes (5B) according to the present invention.

Figure 6 shows that β-1,3/1,6-glucan inhibits the growth of breast cancer (6B) in murine 4T1 breast cancer (6A) and human breast cancer MDA-MB-231, and effectively increases The effect of the therapeutic effect of clinically used chemotherapy drug paclitaxel.

detailed description

Other advantages and features of the present invention will become more apparent from the detailed description of the embodiments of the present invention. It is not limited to the scope of the embodiments.

Example 1

1. Dry the Antarctic brown algae (such as sea velvet, sea bamboo shoots, and Leisong algae), pulverize the algae powder, take 400g of algae powder, add 4000mL of 95% ethanol to the algae powder, soak for 4h, centrifuge and discard the ethanol. Use B The alcohol was repeatedly extracted once and dried to obtain 300 g of defatted algal flour.

2. Take 30 g of the defatted algal flour, add 600 mL of double distilled water, and stir for 2-4 hours at room temperature to obtain an aqueous extract.

3. Centrifuge the supernatant solution; add 600 mL of 3 mol/L calcium chloride aqueous solution to the supernatant, and collect the supernatant after centrifugation, then desalinate with 3000 Da dialysis bag or 1000 Da nanofiltration membrane, concentrate under reduced pressure and freeze. Drying gave 1.6 g of crude polysaccharide.

4. Dissolve 0.5 g of the crude polysaccharide in 2 mL of water, using distilled water and sodium chloride aqueous solution as the mobile phase, and the concentration of sodium chloride aqueous solution is 0.0-2.0 mol/L, using an anion exchange chromatography column (filler is Q Sepharose Fast Flow) Separation, elution with ultrapure water, elution 2 to 8 column volumes. The eluate was collected and concentrated, and then lyophilized to obtain 0.32 g of high-purity β-1,3/1,6-glucan.

The β-1,3/1,6-glucose prepared in this example has a purity of 97.5 wt% and a protein content of 2% wt. High-performance gel permeation chromatography (HPGPC) and eighteen-angle laser scatterometry (MALLS) and differential display are used. The detector (RI) was analyzed in combination and its molecular weight distribution was between 1.1 kDa and 50 kDa.

As shown in Fig. 1, the monosaccharide composition analysis results of the product of the present invention showed that the monosaccharide composition of the obtained polysaccharide had only glucose, indicating that it was glucan.

2, the infrared spectrum of the product of the present invention show, OH stretching vibration of a polysaccharide hydroxyl group 3347cm ^-1, 2917cm ^-1 is the stretching vibration peak of the sugar ring CH, 1646cm ^-1 in a small amount of NH stretching vibration protein The peak, 1371 cm ^-1 is the variable angular vibration peak of CH; 1152 cm ^-1 , 1072 cm ^-1 and 1030 cm ^-1 are the asymmetric stretching vibration peaks of D-glucopyranose ring respectively; 896 cm ^-1 is the characteristic stretching of β-terminal CH The vibration peak, from which it can be judged that the product of the present invention is a β-D-glucopyranose.

Table 1 shows the results of methylation analysis of the products of the present invention. From the data in the table, it is known that Glc (1→, →3) Glc (1→, →6) Glc (1→ and →3,6) Glc (1) exists between each glucose (Glc) in the glucan product of the present invention. → Four kinds of linkage means that the dextran is a dextran with 1→3 linkage as the main chain and 1→6 linkage as the branch.

Table 1 Results of methylation analysis of brown algae β-glucan

Figure 3 is a ¹³ C-NMR spectrum of the product of the present invention. As can be seen from Fig. 3, there is no signal in the range of δ170 to 176×10 ^-6 , indicating that there is no signal between uronic acid and acetyl group in the sample, and δ16～18×10 ^-6 , indicating that there is no methyl sugar in the sample. The signal attribution of the product of the invention is shown in Table 2. From the data in Table 2, the obtained product was β-1,3/1,6-glucose.

¹³ C-NMR signal assignment of β-1,3/1,6 glucan described in Table 2

Example 2

Macrophages (Raw264.7) were cultured in high glucose DMEM medium containing 10% fetal bovine serum, and the cell concentration was adjusted, and the cells were seeded in a 96-well cell culture plate to have a cell concentration of 2 × 10 ⁴ /well. Then, different concentrations of samples were added to make β-1,3/1,6-glucose final concentrations of 5 μg/mL, 25 μg/mL, 50 μg/mL, and 100 μg/mL, respectively, with blank medium as control. After 24 hours of culture, the medium was aspirated, the excess medium was washed with PBS, 0.075% neutral red was added, and the mixture was incubated at 37 ° C, 5% CO ₂ for half an hour, and the excess neutral red was washed with PBS, and cell lysate was added (none Water ethanol: glacial acetic acid = 1:1, volume ratio), after mixing, the absorbance value was measured at 540 nm on the microplate reader, and the sample was evaluated by absorbance to promote the ability of macrophages to phagocytose neutral red. The above experiments were repeated 3 times, and the average was taken for statistical analysis of the results. From the results of Fig. 4, it can be seen that the product of the present invention has a significant promoting effect on macrophage phagocytosis neutral red as the concentration increases from 1 to 100 μg/mL.

Example 3

After the Kunming mice were sacrificed by cervical vertebrae, 75% alcohol was disinfected for 3-5 minutes. The mice were dissected from the ultra-clean table. The spleens of the mice were removed, washed with PBS, ground into individual cells, and counted under a microscope to 4× ^{10 7} cells/mL. The density was inoculated into a 96-well plate. After 4 hours, different concentrations of β-1,3/1,6-glucan (1,5,25, 50, 100 μg/ml) were added, and the administration group was LPS+glucan. Group, ConA + glucan group, LPS + lentinan, ConA + lentinan, LPS final concentration of 20 μg / ml, ConA final concentration of 4 μg / ml. Set 4 parallel holes for each concentration, incubate in a constant temperature incubator with 5% CO2, 37 ° C, and saturated humidity for 72 h. 4 h before the end of the experiment, add 20 μL of 5% MTT to each well, continue to incubate for 4 h, and use a microplate reader. The colorimetric values at 570 nm/630 nm were measured for their respective OD values, and the cell growth rate (RGR) was calculated. The experimental results were repeated 3 times.

The experimental results are shown in Fig. 5. The dextran can effectively increase the responsiveness of spleen lymphocytes to LPS and ConA at a concentration of 1-200 ug/ml, indicating that dextran has a significant effect on promoting spleen lymphocyte proliferation.

Example 4

Mouse Lewis lung cancer cells (LLC) were cultured under appropriate conditions, and when the cells were grown to log phase, the cells were resuspended, counted, inoculated, and passaged in vivo. The mice were subcutaneously inoculated with 1 mm ³ tumor mass in the right forelimb of the right forelimb, and the tumors were grown to a size of about 10 mm ³ . The mice were randomly divided into groups according to their body weight. The high-low dose group of dextran and the positive control of cyclophosphamide (CTX, 30 mg/mL). Group, 10 in each group. The blank group was given 20 mL/kg/day saline. The dose of glucan in the drug-administered group was 1, 4, 10 mg/kg/day, and cyclophosphamide (30 mg/kg/day) was used as the positive control group. 21 days. The tumor weight was measured twice a week and the tumor volume was calculated. After the mice were sacrificed by cervical dislocation, the tumor pieces were taken out and weighed, and the lung tissues were taken out to observe the number of tumor metastatic nodules. The tumor inhibition rate was calculated as follows: tumor inhibition rate (%) = (tumor weight of the blank control group - tumor weight of the treatment group) / tumor weight of the blank control group × 100%; metastasis inhibition rate = (number of nodules in the blank control group) - number of nodules in the treatment group) / number of nodules in the blank control group × 100%, and the results are shown in Tables 3 and 4.

Table 3 Seaweed β-1,3/1,6-glucan inhibited tumor growth in lung cancer-bearing mice (n=10)

Note: x±SE, compared with the model group, **P<0.01, *P<0.05.

As can be seen from Table 3, dextran can effectively inhibit tumor growth, the tumor inhibition rate is 32.9%, and cyclophosphamide has a tendency to lose weight during the course of administration, but the weight of the dextran group does not decrease significantly. It shows that the dextran drug is safe and non-toxic, and has protective effects on animals.

Table 4 Seaweed β-1,3/1,6-glucan inhibited lung cancer metastasis results (n=10)

Note: x±SE, compared with the model group, **P<0.01, *P<0.05.

As can be seen from Table 4, dextran can significantly inhibit the spontaneous lung metastasis of Lewis lung cancer, and the inhibition efficiency is 40.6%, indicating that dextran has an effective inhibitory effect on tumor metastasis and invasion.

Example 5

The mouse breast cancer cell line 4T-1 was resuscitated by liquid nitrogen, and cultured in DMEM medium containing 10% fetal bovine serum; the cultured cells were expanded to reach the desired cell volume, and the cells were digested and collected; 10% fetal bovine was used. The serum DMEM medium was diluted into 5 million/ml cell suspension, and 0.2 ml was inoculated into the right forelimb axillary portion of the mouse after routine disinfection. The diameter of the transplanted tumor was measured with a vernier caliper. After the tumor grew to 100 mm ^{3 , the} mice were randomly divided into groups of 12-15 each; the saline negative control group; the positive control paclitaxel group: paclitaxel 25 mg/kg, paclitaxel 12.5 mg/kg Glucan group: 1 mg/kg, 4 mg/kg; combined drug group was paclitaxel and dextran combination: paclitaxel 12.5 mg/kg +1 mg/kg, paclitaxel 12.5 mg/kg+4 mg/kg. In the mode of administration, paclitaxel was intraperitoneally administered and administered every other day. Glucan is administered intraperitoneally and administered daily. The negative control group was intraperitoneally injected with the same volume of physiological saline, and the results are shown in Tables 5 and 6.

Table 5 Seaweed β-1,3/1,6-glucan inhibited tumor growth in mice bearing breast cancer (

Unit: g)

	Tumor weight	Inhibition rate%
Negative control (saline)	1.08±0.35	/
Paclitaxel 25mg/kg	0.55±0.16	49**
Paclitaxel 12.5mg/kg	0.80±0.25	26*
Dextran 1mg/kg	0.81±0.24	25*
Dextran 4mg/kg	0.87±0.29	19
Paclitaxel 12.5mg/kg+1mg/kg	0.65±0.22	39**
Paclitaxel 12.5mg/kg+4mg/kg	0.60±0.16	44**

Note: x±SE, compared with the model group, **P<0.01, *P<0.05.

The data in Table 5 shows that seaweed β-1,3/1,6-glucan showed significant antitumor effect at 1 mg/kg alone, and the tumor inhibition rate was 25%. After combined with 12.5 mg/kg paclitaxel, 1 and 4 mg/kg of β-1,3/1,6-glucan significantly increased the antitumor effect of paclitaxel, and the tumor inhibition rate of 12.5 mg/kg paclitaxel was 26 % rose to 39% and 44%, indicating that seaweed β-1,3/1,6-glucan has an effect of increasing the antitumor effect of paclitaxel.

Table 6 Seaweed β-1,3/1,6-glucan increased spleen index spleen index of immune organs in mice bearing breast cancer (spleen index = 100 * spleen mass / body weight)

	Spleen index
Negative control (saline)	1.70±0.26
Paclitaxel 25mg/kg	0.52±0.09**
Paclitaxel 12.5mg/kg	1.56±0.3
Dextran 1mg/kg	2.08±0.27**
Dextran 4mg/kg	2.01±0.36*
Paclitaxel 12.5mg/kg+1mg/kg	1.53±0.41
Paclitaxel 12.5mg/kg+4mg/kg	1.32±0.25

Note: x±SE, compared with the model group, **P<0.01, *P<0.05

Lymphocytes of the spleen are mainly divided into T and B lymphocytes and a small amount of NK cells, which complete the cellular and humoral immune functions and the killing effect of tumor cells. The results in Table 6 indicate that the seaweed β-1,3/1,6-glucan alone administration group has a significant effect of increasing the spleen index, P < 0.01; the increase of the spleen index indicates the lymphocyte proliferation of the spleen.

Figure 6A is a visual representation of the size of the tumor obtained during the experiment. The inhibitory effect of glucan on breast tumors was clearly observed, and glucan increased the anti-breast cancer effect of paclitaxel.

Example 6

Liquid nitrogen was used to resuscitate human breast cancer cell line MDA-MB-231, cultured in DMEM medium containing 10% fetal bovine serum; cell fusion reached 80% or more, 0.25% trypsin digestion, passaged at a ratio of 1:3, 4th Cell generation was collected, and 10 ⁷ cells were subcutaneously inoculated into each mouse. When the tumor grew to a diameter of approximately 1-1.5 cm, the tumor tissue was isolated, and the nude mice were inoculated into the armpits with a size of 1 mm ³ by scissors. The diameter of the transplanted tumor was measured with a vernier caliper. After the tumor grew to 10 mm ^{3 , the} mice were randomly divided into groups of 10-12 each; the saline negative control group; the positive control 5-Fu (5-fluorouracil) group: 5 mg/kg Glucan group: 0.5 mg/kg, 1 mg/kg; 5 mg/kg; administered for 21 days. 5-Fu was injected intraperitoneally and administered every other day; administration was given for 21 days. Three doses of dextran were administered intraperitoneally, intraperitoneally, daily for 21 days. The negative control group was intraperitoneally injected with the same volume of normal saline.

The experimental results are shown in Fig. 6B. The algae β-1,3/1,6-glucan has a significant inhibitory effect on the weight of breast cancer tumors in immunodeficient nude mice at different doses, at a low dose of 0.5 mg. The tumor inhibition rate was 25% at /kg/day, and the tumor inhibition rate of breast cancer was 20-25% at doses of 0.5 mg/mg, 1.0 mg/kg, and 5.0 mg/kg.

In summary, when the concentration of the β-1,3/1,6-glucan prepared by the present invention is from 1 to 200 μg/mL, it has the effect of promoting macrophage phagocytosis of neutral red and proliferation of T and B lymphocytes. It is shown to have significant immunopotentiating activity. In vivo mouse experiments showed that at 0.5 mg/kg/day, it significantly inhibited tumor growth and lung tumor metastasis in lung cancer and breast cancer mice, and increased the spleen index of immune organs in tumor-bearing mice. Enhance the potential of anti-tumor drugs.

The above embodiments are only used to illustrate the technical solutions of the present invention, and are not limited thereto; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still Modifications of the technical solutions described, or equivalent replacement of some of the technical features; and such modifications or substitutions do not detract from the nature of the corresponding technical solutions. The spirit and scope of the technical program.

Claims (12)

1. A glucan characterized in that the glucan is β-1,3/1,6-glucan, which has β-1,3-glucose as the main chain and β-1,6- Glucose is branched, and the weight average molecular weight is 1.1 kDa to 50 kDa; the content of glucose in the β-1,3/1,6-glucan is 90 wt% to 98 wt%, and the crude protein content is 0.5% wt-2.5% wt. The rest is moisture.
2. The glucan according to claim 1, wherein the β-1,3/1,6-glucan is extracted from Antarctic brown algae, which is sea velvet, sea bamboo shoot or Leisong. Algae.
3. A method of producing a glucan according to claim 2, which comprises the steps of:

   (1) Degreasing: drying and pulverizing Antarctic brown algae to obtain algal flour, soaking and stirring with an organic solvent to obtain defatted algal flour;

   (2) Water extraction: the degreased algae powder is extracted with water at room temperature to obtain an aqueous extract;

   (3) Classification: The aqueous extract obtained in the step (2) is centrifuged, and an aqueous calcium chloride solution is added to the supernatant obtained by centrifugation; after stirring, the mixture is centrifuged, and the supernatant is taken for dialysis or ultrafiltration desalting, and concentrated under reduced pressure to obtain Crude polysaccharide

   (4) Purification: the crude polysaccharide prepared in the step (3) is dissolved in distilled water, separated by distilled water and sodium chloride aqueous solution, separated and purified by anion exchange resin, detected by phenolic acid method, and the water-eluting component is collected and decompressed. The β-1,3/1,6-glucan was obtained by concentration and lyophilization.
4. The method for preparing a glucan according to claim 3, wherein the ratio of the volume of the organic solvent added in the step (1) to the mass ratio of the algal flour is 10 to 20:1, and degreasing by soaking or intermittent stirring 2 to 8h, the organic solvent used is one or more of ethanol, acetone or methanol.
5. The method for preparing a glucan according to claim 3, wherein the volume of the aqueous solution of calcium chloride added in the step (3) is 1 to 3 times the volume of the supernatant, and the concentration of the aqueous solution of the calcium chloride The membrane used for dialysis or ultrafiltration is 1-3 mol/L, and the molecular weight cut off is 1000 to 3000 Da.
6. The method for preparing a glucan according to claim 3, wherein the concentration of the sodium chloride aqueous solution used in the purification of the anion exchange resin in the step (4) is 0.01 to 2.0 mol/L, and the elution is 2 to 8 columns. volume.
7. Use of the β-1,3/1,6-glucan according to claim 1 or 2 for the preparation of a medicament for immunoenhancement and antitumor medicine and a functional food.
8. Use of the β-1,3/1,6-glucan according to claim 7 for the preparation of a medicament for immunoenhancement and antitumor, and a functional food, characterized in that said β-1,3 When the concentration of /1,6-glucan is from 1 to 100 μg/mL, it has the effect of promoting macrophage phagocytosis of neutral red.
9. Use of the β-1,3/1,6-glucan according to claim 7 for the preparation of a medicament for immunoenhancement and antitumor, and a functional food, characterized in that said β-1,3 When the concentration of /1,6-glucan is 1 to 200 μg/mL, it has an effect of promoting the proliferation of T and B lymphocytes.
10. Use of the β-1,3/1,6-glucan according to claim 7 for the preparation of a medicament for immunoenhancement and antitumor, and a functional food, characterized in that said β-1,3 When the dose of /1,6-glucan is more than 0.5 mg/kg/day, it can effectively inhibit tumor growth and tumor metastasis in tumor-bearing mice.
11. Use of the β-1,3/1,6-glucan according to claim 7 for the preparation of a medicament for immunoenhancement and antitumor, and a functional food, characterized in that said β-1,3 When the dose of /1,6-glucan is more than 0.5mg/kg/day, it can effectively increase the index of immune organs in tumor-bearing mice.
12. Use of the β-1,3/1,6-glucan according to claim 7 for the preparation of a medicament for immunoenhancement and antitumor, and a functional food, characterized in that said β-1,3 /1,6-glucan is compounded with paclitaxel, cisplatin or fluorouracil, and docetaxel for the preparation of antitumor drugs.

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