阅读说明：本技术 浒苔多糖在降低泡沫细胞脂质堆积中的应用 (Application of enteromorpha polysaccharide in reducing foam cell lipid accumulation ) 是由 赵丽华 宋淑亮 陈锚 赵佳锐 朱兰兰 马士玉 关浩 于 2021-09-26 设计创作，主要内容包括：本发明涉及生物技术领域,具体而言,提供了一种浒苔多糖在降低泡沫细胞脂质堆积中的应用。实验发现浒苔多糖可以显著降低泡沫细胞的脂质堆积,促进泡沫细胞恢复成正常巨噬细胞,这不仅扩展了浒苔多糖的用途,也为预防或治疗动脉粥样硬化提供了新的方向。(The invention relates to the technical field of biology, and particularly provides application of enteromorpha polysaccharide in reducing foam cell lipid accumulation. Experiments show that the enteromorpha polysaccharide can obviously reduce the lipid accumulation of foam cells and promote the foam cells to recover to normal macrophages, thereby not only expanding the application of the enteromorpha polysaccharide, but also providing a new direction for preventing or treating atherosclerosis.)

1. The enteromorpha polysaccharide is applied to reducing foam cell lipid accumulation or preparing products for reducing foam cell lipid accumulation for non-disease diagnosis and treatment, and is obtained by a water extraction and alcohol precipitation method.

2. The use according to claim 1, wherein the enteromorpha polysaccharide is used at a concentration of not less than 100 μ g/mL.

3. The use according to claim 2, wherein the enteromorpha polysaccharide is used at a concentration of 800 μ g/mL.

4. The use of claim 1, wherein the step of water extraction and alcohol precipitation comprises: sequentially carrying out ultrasonic water dissolving, filtering and concentrating on the enteromorpha prolifera powder, and adding ethanol to obtain the deposit enteromorpha prolifera polysaccharide.

5. Use according to claim 4, wherein the volume ratio of ethanol to concentrate is 4-6: 1; the volume ratio of the enteromorpha prolifera to the water is 1: 5-10; the volume ratio of water to the concentrated solution is 4-2: 1.

6. use according to claim 4, wherein the ultrasound conditions comprise: 35-45KHz, 55-65 min.

7. Use according to claim 1, characterized in that the reduction of foam cell lipid accumulation is in particular the scavenging of ox-LDL from foam cells.

8. Use according to any of claims 1-7, wherein the product comprises an agent or a medicament.

9. An agent for reducing foam cell lipid accumulation for non-disease diagnostic and therapeutic purposes, comprising enteromorpha polysaccharide for use according to any one of claims 1 to 8.

10. The reagent as claimed in claim 9, wherein the concentration of the Enteromorpha polysaccharide used is 100-800 μ g/mL.

Technical Field

The invention relates to the technical field of biology, and particularly relates to application of enteromorpha polysaccharide in reduction of foam cell lipid accumulation.

Background

The seaweed resources in China are very rich, and the variety of bioactive substances is also many, so that the seaweed biological activity is to be further developed. In recent years, experts at home and abroad are dedicated to the research on the pharmacological function and the biological activity function of the algal polysaccharide, and the seaweed polysaccharide has a great progress and also shows a wide development prospect. Research shows that the algal polysaccharide has the functions of resisting virus, resisting tumor, resisting oxidation, resisting bacteria, diminishing inflammation, etc. The seaweed contains abundant polysaccharide substances, can bring fat out of the body of the chyme, and has good effects of reducing blood fat and cholesterol. The polysaccharide is extracted from the seaweed, and has great significance for marine biopharmaceuticals.

Enteromorpha (Enteromorpha), also called bryophyte and Enteromorpha strip, is an algae plant of Enteromorpha of Ulvaceae of Chlorophyta, mainly grows on gravel of coastal sandy beach, has strong reproductive capacity and can grow in all seasons. The enteromorpha prolifera is rich in various substances beneficial to organisms, has high nutritional value and is algae which is frequently picked by coastal residents in China to eat and be used as feed. Enteromorpha polysaccharide is one of the main active substances of Enteromorpha, and related researches show that the Enteromorpha polysaccharide has various biological effects on organisms, and has the main functions of oxidation resistance, immunoregulation, tumor resistance, virus resistance, growth promotion and the like. The deterioration of the marine ecological environment causes frequent outbreak of enteromorpha in the east coast of China, which causes huge loss in the breeding industry and the tourism industry. Therefore, the enteromorpha prolifera is changed into valuable through research and development and utilization, and the method has important significance for development of related industries and reasonable utilization of resources.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the invention is to provide application of enteromorpha polysaccharide.

It is a second object of the present invention to provide an agent for reducing foam cell lipid accumulation for non-disease diagnostic and therapeutic purposes.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the enteromorpha polysaccharide is applied to reducing foam cell lipid accumulation or preparing products for reducing foam cell lipid accumulation for non-disease diagnosis and treatment, and is obtained by a water extraction and alcohol precipitation method.

Further, the using concentration of the enteromorpha polysaccharide is more than or equal to 100 mu g/mL.

Further, the use concentration of the enteromorpha polysaccharide is 800 mu g/mL.

Further, the water extraction and alcohol precipitation method comprises the following steps: sequentially carrying out ultrasonic water dissolving, filtering and concentrating on the enteromorpha prolifera powder, and adding ethanol to obtain the deposit enteromorpha prolifera polysaccharide.

Further, the volume ratio of the ethanol to the concentrated solution is (4-6): 1; the volume ratio of the enteromorpha prolifera to the water is 1: (5-10); the volume ratio of the water to the concentrated solution is (4-2): 1.

further, the ultrasound conditions include: 35-45KHz, 55-65 min.

Further, reducing foam cell lipid accumulation is specifically the scavenging of ox-LDL within foam cells.

Further, the product comprises an agent or a drug.

An agent for reducing foam cell lipid accumulation for non-disease diagnosis and treatment purposes, which comprises the Enteromorpha polysaccharide in the application.

Further, the use concentration of the enteromorpha polysaccharide is 100-800 mu g/mL.

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

the invention provides application of enteromorpha polysaccharide in reducing foam cell lipid accumulation or preparing products for reducing foam cell lipid accumulation for non-disease diagnosis and treatment. Experiments show that the enteromorpha polysaccharide can obviously reduce the lipid accumulation of foam cells and promote the foam cells to recover to normal macrophages, thereby not only expanding the application of the enteromorpha polysaccharide, but also providing a new direction for preventing or treating atherosclerosis.

The reagent for reducing foam cell lipid accumulation for non-disease diagnosis and treatment purposes can obviously reduce foam cell lipid accumulation and can be used as scientific research application, positive reagent, molding reagent and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is the results of the oil red O staining assay of example 5 for the effect of various concentrations of Enteromorpha polysaccharide in reducing foam cell lipid accumulation (n = 3);

fig. 2 shows the results of the cholesterol kit in example 6 for determining the effect of enteromorpha polysaccharide on reducing foam cell lipid accumulation (n = 3).

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.

The invention provides application of enteromorpha polysaccharide in reducing foam cell lipid accumulation or preparing products for reducing foam cell lipid accumulation for non-disease diagnosis and treatment, wherein the enteromorpha polysaccharide is obtained by a water extraction and alcohol precipitation method.

Experiments show that the enteromorpha polysaccharide can obviously reduce the lipid accumulation of foam cells and promote the foam cells to recover to normal macrophages, thereby not only expanding the application of the enteromorpha polysaccharide, but also providing a new direction for preventing or treating atherosclerosis.

The main extraction method of the enteromorpha polysaccharide comprises a water extraction and alcohol precipitation method, a physical treatment method, an alkali treatment method and an enzyme treatment method. The method adopts a water extraction and alcohol precipitation method, is simple and easy to operate, has low cost and high safety, and does not generate the problem of solvent residue.

In a preferred embodiment, the step of water extraction and alcohol precipitation comprises: sequentially carrying out ultrasonic water dissolving, filtering and concentrating on the enteromorpha prolifera powder, and adding ethanol to obtain the deposit enteromorpha prolifera polysaccharide. The enteromorpha is prepared into powder, ultrasonic water dissolution can improve the dissolution rate of the enteromorpha, the extraction efficiency is improved, and high-temperature heating is avoided. The filtering and concentrating are to remove insoluble impurities, improve the polysaccharide concentration of the enteromorpha and facilitate alcohol precipitation. The concentration is preferably carried out by rotary evaporation. The addition of ethanol precipitates the enteromorpha polysaccharide from the solution by reducing the solubility of the polysaccharide.

Since the influence factors of the extraction efficiency include the material ratio, the ethanol dosage and the like, in a preferred embodiment, the volume ratio of ethanol to the concentrated solution is (4-6): 1; the volume ratio of the enteromorpha prolifera to the water is 1: (5-10); the volume ratio of the water to the concentrated solution is (4-2): 1. in addition, the ultrasonic condition is 35-45KHz, 55-65min, preferably 40KHz, 60 min.

Experiments prove that the enteromorpha polysaccharide can remove ox-LDL in foam cells, so that the lipid accumulation of the foam cells is reduced. With the increase of the concentration of the enteromorpha polysaccharide, the capacity of the enteromorpha polysaccharide for reducing the lipid accumulation of the foam cells is gradually enhanced, and when the concentration of the enteromorpha polysaccharide is 800 mu g/mL, the enteromorpha polysaccharide can remarkably reduce the lipid accumulation of the foam cells, and the degree can reach 51.1%.

The product prepared by using the enteromorpha polysaccharide as a main active ingredient, such as a reagent or a medicament, has the function of reducing the accumulation of foam cell lipid. The invention provides related reagents which are used for scientific research purposes, positive reagents, molding reagents and the like.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

Example 1: extraction of enteromorpha polysaccharide

Cleaning 1kg of enteromorpha prolifera, drying, crushing into powder by using a high-speed traditional Chinese medicine crusher, adding 10 times of distilled water by volume, and performing ultrasonic water bath (40KHz for 60min) to obtain enteromorpha prolifera liquid. Standing overnight, filtering to remove insoluble precipitate, concentrating the filtrate to one fourth of the original volume by rotary evaporation, adding 5 times volume of ethanol into the concentrated solution, standing overnight, centrifuging at 4000rpm for 20min to obtain precipitate, washing with anhydrous ethanol for three times to remove small molecular substances, centrifuging at 4000rpm for 10min, removing ethanol to obtain wet product, and oven drying to obtain the final product.

Example 2: extraction of enteromorpha polysaccharide

Cleaning 1kg of enteromorpha prolifera, drying, crushing into powder by using a high-speed traditional Chinese medicine crusher, adding distilled water with the volume 5 times that of the powder, and performing ultrasonic water bath (40KHz for 60min) to obtain enteromorpha prolifera liquid. Standing overnight, filtering to remove insoluble precipitate, concentrating the filtrate to one fourth of the original volume by rotary evaporation, adding 5 times volume of ethanol into the concentrated solution, standing overnight, centrifuging at 4000rpm for 20min to obtain precipitate, washing with anhydrous ethanol for three times to remove small molecular substances, centrifuging at 4000rpm for 10min, removing ethanol to obtain wet product, and oven drying to obtain the final product.

Example 3: extraction of enteromorpha polysaccharide

Cleaning 1kg of enteromorpha prolifera, drying, crushing into powder by using a high-speed traditional Chinese medicine crusher, adding 10 times of distilled water by volume, and performing ultrasonic water bath (40KHz for 60min) to obtain enteromorpha prolifera liquid. Standing overnight, filtering to remove insoluble precipitate, concentrating the obtained filtrate to half of the original volume in a rotary evaporation mode, adding 5 times of ethanol by volume into the concentrated solution, standing overnight, centrifuging at 4000rpm for 20min to obtain precipitate, washing with anhydrous ethanol for three times to remove small molecular substances, continuously centrifuging at 4000rpm for 15min, discarding ethanol to obtain wet product, and oven drying to obtain the final product.

The results of statistics of the weight of the enteromorpha polysaccharide obtained in examples 1 to 3 are shown in table 1.

Table 1 results of polysaccharide extraction from enteromorpha (n = 3)

The results show that the polysaccharide extraction rate of example 1 is higher.

Example 4: determination of total sugar content and sulfate radical content of enteromorpha polysaccharide

The total sugar content of the enteromorpha polysaccharide is determined by adopting an SN/T4260-:

preparation of reagents: concentrated sulfuric acid; 80% ethanol solution; glucose; 80% phenol solution (weighing 80 g phenol in 100 mL beaker, adding water to dissolve, transferring to 100 mL brown volumetric flask for constant volume, and storing in 4 ℃ refrigerator in dark place); 5% phenol solution (5 mL of 80% phenol solution is absorbed and dissolved in 75 mL of water, and the mixture is mixed uniformly and prepared on site); 100 mg/L standard glucose solution (0.100 g glucose is weighed into a 100 mL beaker and dissolved in water to a volume of 1000 mL).

Sample preparation: dissolving 10 mg of enteromorpha polysaccharide to be detected in 5mL of pure water to prepare 2000 mu g/mL solution, fully dissolving overnight at 4 ℃, and placing 1mL in a 20mL test tube with a plug to be detected.

Preparation of a standard curve: 0mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL of standard glucose working solution is respectively sucked and placed in a 20mL test tube with a plug, and the solution is supplemented to 1.0 mL by distilled water and is to be tested.

And (3) determination: 1.0 mL of 5% phenol solution was added to the test solution, 5.0 mL of concentrated sulfuric acid was added rapidly, the mixture was left to stand for 10min, mixed thoroughly with a vortex shaker, and the test tube was placed in a 30 ℃ water bath to react for 20 min. And measuring the absorbance at 490 nm of an ultraviolet spectrophotometer, and calculating a standard curve and the total sugar content in the sample by taking the glucose concentration as an abscissa and the absorbance as an ordinate.

The sulfate radical content in the enteromorpha polysaccharide is measured by adopting a GB13025891 barium chloride gelatin method:

preparation of reagents: 0.5% gelatin (0.5 g gelatin dissolved in 100 mL pure water at 60-70 deg.C in water bath, fully dissolved for 12h, ultrafiltered with 0.45 um ultrafiltration membrane); barium chloride-gelatin solution (0.5 g barium chloride dissolved in 100 mL 0.5% gelatin, stirred well, stored at 4 ℃ for use); potassium sulfate standard solution (68 mg potassium sulfate dissolved in pure water, constant volume to 100 mL); 500 mL of 1M nitric acid solution.

Preparation of a standard curve: respectively measuring 0mL, 2 mL, 4mL, 6mL, 8mL, 10 mL, 12 mL and 14mL of prepared potassium sulfate solution, and fixing the volume to 25 mL by using 1M nitric acid to be detected.

Sample preparation: respectively taking 10 mg of enteromorpha polysaccharide to be detected, dissolving in 5mL of pure water to prepare 2000 ug/mL solution, and fully dissolving overnight at 4 ℃.

And (3) determination: 2 mL of the solution to be detected is put into a test tube with a plug respectively, 5mL of concentrated nitric acid is added, and acidolysis is carried out in boiling water bath for 180 min. The volume of the acidolysis solution is determined to be 10 mL, 2 mL of the acidolysis solution is respectively taken, 3mL of barium chloride-gelatin solution is respectively added into each tube, and the mixture is fully shaken up. Measuring absorbance at 360 nm with ultraviolet spectrophotometer to obtain sulfate radical concentration [ SO₄ ^2-]Calculating the standard curve and [ SO ] in the sample by using the abscissa and the ordinate as the absorbance₄ ^2-]。

Table 2 total sugar content and sulfate content of enteromorpha polysaccharide (n = 3)

The results show that the total sugar content and the sulfate radical content of the enteromorpha polysaccharide prepared in the examples 1, 2 and 3 are relatively close.

Example 5: determination of effect of enteromorpha polysaccharide on reducing foam cell lipid accumulation by oil red O method

The density is 5 multiplied by 10⁴one/mL RAW264.7 cells were seeded in 24-well plates at 37 ℃ in 5% CO₂After further incubation for 24h in the environment, ox-LDL was administered at 80. mu.g/mL, and after incubation for 24h, various concentrations (0. mu.g/mL, 100. mu.g/mL, 200. mu.g/mL) were addedμ g/mL, 400 μ g/mL and 800 μ g/mL) of enteromorpha polysaccharide for 24h, followed by lipid droplet staining using oil red O.

The results are shown in FIG. 1. The result shows that the content of lipid droplets in the foam cells is gradually reduced along with the increase of the concentration of the enteromorpha polysaccharide.

Example 6: method for determining effect of enteromorpha polysaccharide on reducing foam cell lipid accumulation by using cholesterol ester content kit

The density is 5 multiplied by 10⁴one/mL RAW264.7 cells were seeded in 24-well plates at 37 ℃ in 5% CO₂After the culture is continued for 24h in the environment, 80 mu g/mL ox-LDL is given, after the incubation is performed for 24h, Enteromorpha polysaccharide with different concentrations (0 mu g/mL, 100 mu g/mL, 200 mu g/mL, 400 mu g/mL and 800 mu g/mL) is added for further incubation for 24h, and then the content determination of cholesterol ester is performed by using a cholesterol kit.

The results are shown in fig. 2, and the capacity of the enteromorpha polysaccharide for reducing the accumulation of foam cell lipid is gradually strengthened along with the increase of the concentration of the enteromorpha polysaccharide. When the concentration of the enteromorpha polysaccharide is 800 mu g/mL, the enteromorpha polysaccharide can remarkably reduce the accumulation of foam cell lipid, and the degree can reach 23.7%.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.