CN117247472B

CN117247472B - Fucoidin and preparation method and application thereof

Info

Publication number: CN117247472B
Application number: CN202311186354.5A
Authority: CN
Inventors: 马慧; 吴科锋; 赵奇; 邓路铭; 叶华
Original assignee: Guangdong Zhanjiang Institute Of Marine Medicine; Guangdong Medical University
Current assignee: Guangdong Zhanjiang Institute Of Marine Medicine; Guangdong Medical University
Priority date: 2023-09-14
Filing date: 2023-09-14
Publication date: 2024-03-19
Anticipated expiration: 2043-09-14
Also published as: CN117247472A

Abstract

The invention belongs to the technical field of biological polysaccharide, and particularly relates to brown algae polysaccharide, and a preparation method and application thereof. The fucoidan is an isopolysaccharide polymerized by D-glucose, and consists of alpha-D-Glcp- (1- & gt, 4- & gt) -alpha-D-Glcp- (1- & gt and 4, 6) -alpha-D-Glcp- (1- & gt, wherein the molar ratio of the alpha-D-Glcp to the 1- & gt is 5:87.5:7.5.

Description

Fucoidin and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological polysaccharide, and particularly relates to brown algae polysaccharide, and a preparation method and application thereof.

Background

Osteoporosis is a systemic metabolic bone disease characterized by low bone mass, deterioration of bone tissue microstructure, increased bone fragility, and susceptibility to fracture. With the rapid increase in the world's aging population, osteoporosis has become a significant public health problem. An imbalance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption is considered to be a major cause of osteoporosis. At present, clinical anti-osteoporosis medicines are mainly divided into two types: anti-absorption drugs (bisphosphonates, selective estrogen response modifiers, monoclonal antibodies, etc.) and anabolic drugs (human parathyroid hormone analogues, anti-sclerostin monoclonal antibodies, etc.). However, these drugs have their own limitations or various side effects in long-term use. For example, continued estrogen replacement therapy can lead to an increased risk of breast cancer, stroke, and heart disease, while prolonged use of bisphosphonates can lead to atypical femoral fractures and jawbone necrosis. Therefore, there is a need to find effective components with less side effects from natural resources to prevent and treat osteoporosis.

In China, several Chinese medicinal materials have long been used for preventing and treating osteoporosis, such as radix Morindae officinalis, achyranthis radix, radix Angelicae sinensis, etc. Polysaccharides are one of the main active and functional components of these traditional Chinese medicines, some of which show good anti-osteoporosis effects by balancing bone resorption and bone formation. Sargassum has been used as a marine-derived traditional Chinese medicine for thousands of years in the treatment of tumors, scurvy, oedema, beriberi and chronic bronchitis. Polysaccharides isolated from brown algae have not only been found to be safe but have a wide range of biological activities such as enhancing immunity, antioxidant, antitumor, anti-aging, anticoagulant, antiviral and antibacterial activities.

Therefore, the invention hopes to develop further research on brown algae polysaccharide so as to obtain a polysaccharide functional component with good treatment effect on osteoporosis.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides brown algae polysaccharide, and a preparation method and application thereof. The brown algae polysaccharide is an isopolysaccharide polymerized by d-glucose, has obvious effects of promoting cell osteogenic differentiation and inhibiting cell adipogenic differentiation, and has wide application prospect in the treatment of osteoporosis.

The invention provides brown algae polysaccharide, which has a structural formula shown in a formula (1):

the brown seaweed polysaccharide is an isopolysaccharide formed by polymerizing d-glucose, and consists of alpha-d-Glcp- (1- & gt, 4- & gt) -alpha-d-Glcp- (1- & gt and 4, 6) -alpha-d-Glcp- (1- & gt, wherein the molar ratio of the alpha-d-Glcp to the 4, 6) -alpha-d-Glcp- (1- & gt is 5:87.5:7.5, and the average molecular weight of the brown seaweed polysaccharide is 27476g/mol and the number average molecular weight is 20588g/mol.

The invention also provides a preparation method of the brown algae polysaccharide, which comprises the following steps:

s1, drying, crushing, water extraction and alcohol precipitation of brown algae to obtain brown algae crude polysaccharide SFP;

s2, dissolving the brown algae crude polysaccharide SFP in water, centrifuging to obtain supernatant, and purifying on a DEAE-52 cellulose column to obtain a polysaccharide component;

s3, eluting the polysaccharide component on a gel purification system column sequentially by adopting NaCl solution and water as fluidity to obtain the brown algae polysaccharide.

Preferably, the brown algae is gulfweed.

Preferably, in the step S1, the water extraction temperature is 63-68 ℃, the number of times of water extraction is 2-4, and the time of each water extraction is 3-6h.

Preferably, in the step S1, the operation of alcohol precipitation is: mixing the concentrated extract with absolute ethanol at a ratio of 1:4, mixing the materials according to the volume ratio, and carrying out alcohol precipitation.

Preferably, in the step S1, the step of deproteinizing and freeze-drying is further included after the step of performing the alcohol precipitation.

More preferably, the deproteinizing is performed using a Sevage reagent.

Preferably, the elution is performed with water, 0.2M NaCl solution, 0.4M NaCl solution, 0.6M NaCl solution, 0.8M NaCl solution, 1.0M NaCl solution, in this order in step S2.

Preferably, the concentration of the NaCl solution in step S3 is 0.3M.

The invention also provides application of the brown algae polysaccharide in preparing a medicament for promoting cell osteogenic differentiation and adipogenesis inhibition, wherein the concentration of the brown algae polysaccharide is 12.5-50 mug/mL.

The invention also provides application of the brown algae polysaccharide in preparing a medicament for treating/preventing osteoporosis. The brown algae polysaccharide has the functions of promoting cell osteogenic differentiation and inhibiting cell adipogenic differentiation, and can be used for treating/preventing osteoporosis and other related diseases.

Compared with the prior art, the invention has the following beneficial effects:

the invention develops researches on polysaccharide components in brown algae, discovers and purifies the polysaccharide components with remarkable effects of promoting cell osteogenic differentiation and inhibiting cell adipogenic differentiation, can be used as a safe medicine for treating and preventing osteoporosis, and has good application prospect.

Drawings

FIG. 1 shows the results of gas chromatography-mass spectrometry analysis of fucoidan SFP-1 (red) and monosaccharide standard (black) (1: rhamnose; 2: caramel; 3: arabinose; 4: xylose; 5: mannose; 6: glucose; 7: galactose);

FIG. 2 is a FT-IR spectrum of fucoidan SFP-1;

FIG. 3 is an immunofluorescence staining image of fucoidan SFP-1 on promotion of osteogenic differentiation of C3H10 cells;

FIG. 4 is an immunofluorescence staining image of fucoidan SFP-1 on inhibiting adipogenic differentiation of C3H10 cells;

FIG. 5 is a fluorescent image of fucoidan SFP-1 promoting osteogenic mineralization in zebra fish.

Detailed Description

In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples will be presented. It should be noted that the following examples do not limit the scope of the invention.

The starting materials, reagents or apparatus used in the following examples are all available from conventional commercial sources or may be obtained by methods known in the art unless otherwise specified.

Example 1

Preparation of fucoidan SFP-1

The brown algae polysaccharide SFP-1 is prepared by adopting the following preparation method:

(1) Drying and crushing the cleaned gulfweed to obtain brown algae powder; degreasing and decoloring brown algae powder with 95% ethanol (v/v), airing, and adding deionized water with the volume of 20 times to obtain a feed liquid mixture;

(2) Extracting crude polysaccharide from the feed liquid mixture at 65deg.C for 4 hr for 3 times; mixing the extractive solutions, and concentrating by evaporation;

(3) Mixing the extract obtained after evaporation concentration with absolute ethyl alcohol according to a ratio of 1:4, mixing the materials in a volume ratio, and carrying out alcohol precipitation at 5 ℃; removing protein by Sevage method, and freeze drying the precipitate to obtain brown algae crude polysaccharide SFP;

(4) Redissolving the obtained brown algae crude polysaccharide SFP in distilled water, centrifuging at 4000r/min for 15min, and collecting supernatant; the supernatant was applied to a DEAE-52 cellulose column (1.6 cm. Times.20 cm. In this order), eluted with distilled water, 0.2M, 0.4M, 0.6M, 0.8M, 1.0M NaCl solution at a constant flow rate of 0.3mL/min, and the eluate was collected and monitored at 490 nm by the phenol-sulfuric acid method. The polysaccharide fraction was collected and concentrated to dryness.

(5) The polysaccharide fraction was eluted on a gel purification system column (1.6 cm. Times.100 cm. In another example) with 0.3M NaCl solution as mobile phase; further purifying with distilled water as mobile phase on gel purification system (1.6 cm×100 cm) to obtain fucoidan SFP-1.

FIG. 1 shows the results of gas chromatography-mass spectrometry analysis of fucoidan SFP-1 (red) and monosaccharide standard (black), and shows that single and symmetrical elution peaks appear in the eluate, indicating fucoidan SFP-1 is an isopolysaccharide composed of glucose. The average molecular weight of the purified fucoidan SFP-1 was 27476g/mol as further determined by GPC-MALS-RI. In addition, SFP-1 was also determined to have a number average molecular weight (Mn) of 20588g/mol and Mw/Mn, i.e., polydispersity index (PDI), of 1.33, indicating that it has polydispersity in molecular size.

Example 2

Structural characterization of fucoidan SFP-1

FT-IR spectroscopy can predict polysaccharide structure based on specific functional groups of the polysaccharide. FIG. 2 shows the FT-IR spectrum of fucoidan SFP-1 obtained in example 1. As can be seen from FIG. 2, at 3376.75cm ⁻¹ There is a broad and strong stretching peak, which is the stretching vibration absorption peak of hydroxyl, and the region is the characteristic absorption peak of the polysaccharide. At 2927.41cm ^-1 And 1419.35cm ⁻¹ The absorption bands at the sites are the tensile and flexural vibration absorption peaks of polysaccharide C-H, respectively. At 1639.20cm ⁻¹ The weak absorption band occurs here due to the absorption of water. In addition, the peak value of the stretching vibration is 1157.08cm ⁻¹ ~1035.59cm ⁻¹ The peak between the peaks is caused by stretching vibration of C-O, at 927.59cm ⁻¹ The characteristic absorption band at this point indicates the presence of a pyranose ring.

Monosaccharide composition and type of glycosidic bond

The type and proportion of glycosyl bonds of fucoidan SFP-1 were determined by methylation analysis. The methylation analysis results of GC-MS of fucoidan SFP-1 are shown in Table 1. These results indicate that SFP-1 is a homoglycan consisting of three components, α -d-GlcP- (1→, →4) - α -d-GlcP- (1→and 4, 6) - α -d-GlcP- (1→the molar ratio of the three components is 5:87.5:7.5.

TABLE 1 methylation analysis of fucoidan SFP-1

Methylated sugar	Connection type	Molar ratio of	Mass fraction (m/z)
				1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-D-glucitol	d-Glcp-1→	5	43,71,87,101,117,129,145,161,205
1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl-D-glucitol	→4)-d-Glcp-(1→	87.5	43,87,99,101,113,117,129,131,161,173,233
				1,4,5,6-tetra-O-acetyl-2,3-di-O-methyl-D-glucitol	→4,6)-d-Glcp-(1→	7.5	43,71,85,87,99,101,117,127,159,161,201

Nuclear magnetic resonance analysis

Through brown algae polysaccharideNuclear magnetic profile analysis of SFP-1: (A) ¹ H-NMR、(B) ¹³ C-NMR、(C) ¹ H- ¹ H COSY、(D) ¹ H- ¹³ C HSQC、(E) ¹ H- ¹³ C HMBC、(F) ¹ H- ¹ H NOESY, the nuclear magnetic characterization result of fucoidan SFP-1 can be obtained (shown in Table 2).

TABLE 2 Nuclear magnetic characterization results of fucoidan SFP-1

Chemical structure

According to analysis, the structural formula of the fucoidan SFP-1 in the invention is shown as follows:

example 3

Fucoidan SFP-1 research on promotion of osteogenic differentiation of C3H10 cells (mouse mesenchymal stem cell line)

Experimental grouping:

SFP-1 treatment group: the concentration of the fucoidan SFP-1 is respectively set to be 12.5, 25 and 50 mug/mL, and a control group is set at the same time;

the experimental steps are as follows:

C3H10 cells were taken in 24 well plates at 3.0X10 ⁵ Cell/well density culture for 24h. Subsequently, the medium was changed to osteoinductive medium (supplemented with 100 nM meters dexamethasone, 50. Mu.M ascorbic acid, 10 mM. Beta. -glycerophosphate, 10% fetal bovine serum, and 1% antibiotics) and SFP-1 (0, 12.5, 25, 50. Mu.g/mL) was added at various concentrations. After 11 days of treatment, the differentiation capacity of C3H10 cells was examined by alkaline phosphatase (ALP) staining and the mineralization capacity of C3H10 cells was examined by alizarin red S staining. The effect of SFP-1 on the osteogenic differentiation of C3H10 cells was examined using a stereoscope (visualization of stained cells, analysis of images using imageJ software. Immunofluorescence).

Experimental results:

as shown in FIG. 3, SFP-1 treated groups significantly increased the number of fluorophores and fluorescence intensity and were dose dependent compared to control groups after staining with fluorescence labelled anti-sparc. These results suggest that fucoidan SFP-1 can significantly promote osteogenic differentiation of C3H10 cells.

Example 4

Fucoidan SFP-1 research on promotion of lipid differentiation of C3H10 cells (mouse mesenchymal stem cell line)

Experimental grouping:

the experimental steps are as follows:

C3H10 cells were taken at 5.0X10 ⁴ Cell/well density was seeded in 24-well plates and incubated in DMEM for 24 hours. Subsequently, C3H10 cells were cultured with adipogenesis inducing groups (rosiglitazone 5 mmol/L, insulin 4.5. Mu.g/mL, 3-isobutyl-1-methylxanthine 11.5 mg/mL, dexamethasone 2 mmol/L) and fucoidan SFP-1 (0, 12.5, 25, 50 mg/mL) was added at various concentrations. After 8 days of culture, the adipogenic differentiation ability of C3H10 cells was evaluated by the ORO staining method. Stained cells were observed with a stereoscope (SZN-71, SOPTOP, china) and the images were analyzed with imageJ software.

Experimental results:

the effect of fucoidan SFP-1 on adipogenic differentiation of C3H10 cells was evaluated by ORO staining, and the results of FIG. 4 show that the dark red area after ORO staining showed the presence of adipogenic differentiation. The SFP-1 treated group significantly inhibited the adipogenic differentiation capacity of C3H10 cells compared to the control group and was dose dependent. These results suggest that fucoidan SFP-1 can inhibit adipogenic differentiation of C3H10 cells.

Example 5

Fucoidan SFP-1 promotes osteogenic mineralization in zebra fish body

Experimental grouping:

the experimental steps are as follows:

zebra fish male and female embryos are used simultaneously, and the synchronized embryos are stored in 24 well plates 3 days after fertilization. According to the cell test dose, the zebra fish larvae were treated with brown algae SFP-1 (12.5, 25, 5, 50. Mu.g/mL) at different concentrations, and the zebra fish larvae treated with egg water were used as a control group.

For each treatment, 30 zebra fish larvae were used. Zebra fish larvae were collected 11 days after fertilization and then soaked in 0.2% calcein solution for 15 min. The zebra fish larvae were then rinsed thoroughly 3 times (5 min/time) in egg water, anesthetized with MS-222, and mounted on a lower ballast slide. The skull of zebra fish was scanned using a Scanning Disk Confocal Microscope (SDCM) and the fluorescence images were quantitatively analyzed using ImageJ.

Experimental results:

the fluorescence image of zebra fish skull after fertilization for 11 days is shown in fig. 5. When the zebra fish larvae were exposed to fucoidan SFP-1 (12.5, 25, and 50. Mu.g/mL), the number of stained mineralized tissues increased with increasing fucoidan SFP-1 concentration. The SFP-1 treated group significantly increased the relative fluorescence intensity of the zebra fish skull in a dose-dependent manner. These results further confirm the effect of fucoidan SFP-1 in promoting osteogenic mineralization in vivo.

In conclusion, the fucoidan SFP-1 obtained by separation and purification of DEAE-52 cellulose and gel purification system column has promotion effect on osteogenesis mineralization and inhibition effect on adipogenesis differentiation, and the fucoidan has good safety, so that the fucoidan can be used as a medicament for treating osteoporosis with outstanding potential.

The embodiments of the present application have been described in detail above with reference to the accompanying drawings, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present application.

Claims

1. The brown algae polysaccharide is characterized in that the structural formula of the brown algae polysaccharide is shown as a formula (1):

；

（1）

the brown seaweed polysaccharide is formed by polymerizing glucose, and the saccharide units comprise alpha-d-Glcp- (1- & gt4) -alpha-d-Glcp- (1- & gt4, 6) -alpha-d-Glcp- (1- & gt) and the molar ratio is 5:87.5:7.5 respectively through methylation analysis;

the average molecular weight of the fucoidan is 27476g/mol.

2. The method for preparing fucoidan according to claim 1, comprising the steps of:

s3, eluting the polysaccharide component on a gel purification system column sequentially by adopting NaCl solution and water as fluidity to obtain the brown algae polysaccharide;

the brown algae is Sargassum;

in the step S1, the temperature of water extraction is 63-68 ℃;

in the step S1, the alcohol precipitation is performed as follows: mixing the concentrated extract with absolute ethanol at a ratio of 1:4, mixing the materials according to the volume ratio for alcohol precipitation;

the elution is performed with water, 0.2M NaCl solution, 0.4M NaCl solution, 0.6M NaCl solution, 0.8M NaCl solution, 1.0M NaCl solution in this order in step S3.

3. The method according to claim 2, wherein in the step S1, the number of water extractions is 2 to 4, and the time of each water extraction is 3 to 6 hours.

4. The use of fucoidan according to claim 1 for the preparation of a medicament for promoting cell osteogenic differentiation and adipogenesis inhibition, wherein the fucoidan has a concentration of 12.5-50 μg/mL.

5. The use of fucoidan according to claim 1 for the preparation of a medicament for the treatment/prevention of osteoporosis.