Application of seaweed oligosaccharide in preparation of product for adjuvant treatment of Parkinson's disease

Abstract

The invention belongs to the field of seaweed product application, and particularly relates to application of seaweed oligosaccharide in preparation of a product for adjuvant treatment of Parkinson's disease. According to the method, a seaweed oligosaccharide component is obtained through biotransformation of probiotics, and the seaweed oligosaccharide capable of assisting in treating Parkinson's disease is obtained through pH regulation, ion exchange chromatography and gel chromatography separation and purification. The extraction liquid is refined and purified through DEAE SephadexTMA-25 column chromatography and Bio-Gel P-2 gel column chromatography, the purity of the obtained seaweed oligosaccharide is improved, and the purity of the seaweed oligosaccharide prepared through the method reaches up to about 93%. The seaweed oligosaccharide obtained by the method has a good effect on treatment of Parkinson's disease, and a novel safe product is provided for adjuvant treatment of Parkinson's disease.

Description

Technical Field

[0001] The invention belongs to the application field of seaweed functional products, and specifically relates to the application of seaweed oligosaccharides in preparing products for auxiliary treatment of Parkinson's disease. Background Art

[0002] Seaweed oligosaccharides are small molecular fragments formed by enzymatic degradation of seaweed polysaccharides. Compared with seaweed polysaccharides, they have the characteristics of good water solubility, small molecular weight, and are more easily absorbed and utilized by organisms.

[0003] Parkinson's disease is a neurodegenerative disease caused by the selective loss of dopaminergic neurons. It is common in middle-aged and elderly people. With the progress of aging, the incidence of Parkinson's disease is increasing year by year. There is no effective method for treating Parkinson's disease in the existing technology, and most of the existing drugs can only temporarily relieve symptoms, but cannot prevent or slow down the progression of the disease.

[0004] At present, the research on seaweed oligosaccharides is mostly focused on the preparation method of seaweed oligosaccharides, and is committed to increasing the content of seaweed oligosaccharides so that the prepared seaweed oligosaccharide products contain more active ingredients. However, there are few disclosures on the efficacy of active ingredients in seaweed oligosaccharides, and there are few disclosures on the structure-activity relationship of seaweed oligosaccharides structure and function. There are even fewer relevant literature disclosures on the mechanism between seaweed oligosaccharides and Parkinson's disease. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a new use of seaweed oligosaccharides in the preparation of Parkinson's auxiliary treatment products. Seaweed oligosaccharide components are obtained by probiotic biotransformation, and seaweed oligosaccharides with specific structures are obtained by separation and purification. The seaweed oligosaccharides with this structure can be used in the preparation of Parkinson's auxiliary treatment products, providing a new, effective and safe product for the auxiliary treatment of Parkinson's disease.

[0006] The technical solution of the present invention is as follows: The present invention provides an application of seaweed oligosaccharide in preparing a product for assisting the treatment of Parkinson's disease. The seaweed oligosaccharide has the following structure: .

[0007] Preferably, seaweed oligosaccharides are obtained by the following preparation method: the cleaned and dried seaweed is crushed, enzymolyzed, fermented, the fermented supernatant is evaporated and concentrated, then alcohol extracted, the alcohol supernatant is concentrated under reduced pressure, purified, and freeze-dried to obtain the seaweed oligosaccharide product.

[0008] The seaweed is selected from any one of wakame, kelp, hijiki, sargassum and kelp.

[0009] Preferably, cellulase and pectinase are used for enzymatic hydrolysis; the added weight of cellulase accounts for 1-3% of the weight of the seaweed raw material, and the added weight of pectinase accounts for 0.5-3% of the weight of the seaweed raw material.

[0010] Preferably, during fermentation, aerobic fermentation is first performed using Saccharomyces cerevisiae, and then anaerobic fermentation is performed using Lactobacillus plantarum and Lactobacillus rhamnosus. The inoculation amounts of the Saccharomyces cerevisiae, Lactobacillus plantarum and Lactobacillus rhamnosus are all 2.5-3.5% of the volume of the raw material to be fermented.

[0011] Preferably, the alcohol used in the alcohol extraction is anhydrous ethanol, and the specific operation is: adding anhydrous ethanol to the concentrated fermentation supernatant until the final volume fraction of anhydrous ethanol is 60-80%.

[0012] Preferably, the purification steps are as follows: The alcohol supernatant was concentrated under reduced pressure and dried. The extract was re-dissolved in distilled water, and the pH was adjusted to 7-8 with sodium hydroxide solution. The precipitate was removed by centrifugation. The supernatant was placed in a 500D dialysis bag, dialyzed with deionized water for 24 hours, and then concentrated. DEAE Sephadex was added. TM The A-25 chromatography column was eluted with pure water, and the eluate was collected. The eluate was concentrated and then eluted with the gel column Bio-Gel P-2, and the eluate was collected in sections.

[0013] In the present invention, after the seaweed is subjected to enzymatic fermentation treatment, ethanol is used to elute the supernatant obtained by fermentation, which is beneficial for dissolving effective ingredients such as oligosaccharides in the alcohol supernatant, removing impurities such as macromolecular proteins and polysaccharides, and facilitating further separation and purification.

[0014] Preferably, the Parkinson's disease auxiliary treatment product includes any one of oral liquid, compressed candy, capsule, solid granule, and solid beverage.

[0015] Preferably, the molecular weight of seaweed oligosaccharide is 1031 Da.

[0016] The beneficial effects of the present invention are: (1) The present invention first uses pH control to crudely purify the fermentation broth after ethanol extraction, and then uses DEAE Sephadex TM The extract was refined and purified by A-25 column chromatography and Bio-Gel P-2 gel column chromatography to improve the purity of the obtained seaweed oligosaccharides. The purity of the seaweed oligosaccharides obtained by this method was as high as about 93%, which was much higher than the product in the comparative example; (2) The present invention obtains seaweed oligosaccharide components through probiotic biotransformation, and obtains seaweed oligosaccharides with a specific structure through separation and purification. The seaweed oligosaccharides with this structure have a good effect on the auxiliary treatment of Parkinson's disease, providing a new product for the auxiliary treatment of Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the HPLC peak diagram of the seaweed oligosaccharide prepared in Example 1 of the present invention; Figure 2 This is the ion flow diagram of N-acetylglucosamine mannopentose (Man5GlcNAc-I) in the seaweed oligosaccharide prepared in Example 1 of the present invention; Figure 3 The effects of different treatments of the present invention on cell survival rate in Parkinson's cell model; Figure 4 This is a high performance liquid chromatogram of seaweed oligosaccharides obtained by the method in Comparative Example 1 of the present invention; Figure 5 This is a high performance liquid chromatogram of seaweed oligosaccharides obtained by the purification method in Comparative Example 2 of the present invention; Figure 6 This is a high performance liquid chromatogram of seaweed oligosaccharides obtained by the purification method in Comparative Example 3 of the present invention; Figure 7 This is a high performance liquid chromatogram of seaweed oligosaccharides obtained by the purification method in Comparative Example 4 of the present invention. DETAILED DESCRIPTION

[0018] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific implementation methods.

[0019] In the following embodiments, the brewer's yeast used in the fermentation process was deposited in the China Center for Type Culture Collection on November 29, 2021, address: Wuhan University, Wuhan, China, with a collection number of: CCTCC NO: M 20211499, and a classification name of: Saccharomyces cerevisiae AMnb091.

[0020] Lactobacillus plantarum was deposited in the China Center for Type Culture Collection on November 29, 2021, with the address: Wuhan University, Wuhan, China. The deposit number is: CCTCC NO: M 20211500, and the classification name is: Lactobacillus plantarum LP1406 Lactiplantibacillus plantarum LP1406.

[0021] Lactobacillus rhamnosus was deposited in the China Center for Type Culture Collection on October 21, 2022, address: Wuhan University, Wuhan, China, with the deposit number: CCTCC NO: M 20221626, and the classification name: Lactobacillus rhamnosusF-B4-1.

[0022] Example 1 The preparation method of seaweed oligosaccharide comprises the following steps: (1) Enzymatic hydrolysis: The cleaned and dried kelp was crushed, and the obtained seaweed powder was enzymatically hydrolyzed with cellulase and pectinase. The added weight of cellulase accounted for 2.5% of the weight of the seaweed powder, and the added amount of pectinase accounted for 0.5% of the weight of the seaweed powder. The conditions of enzymatic hydrolysis were: enzymatic hydrolysis at 55°C for 2 h; after enzymatic hydrolysis, the enzyme was inactivated at 95°C for 10 min. (2) Fermentation: aerobic fermentation is performed using Saccharomyces cerevisiae AMnb091, and then anaerobically fermenting the enzymatic hydrolysate after the enzyme inactivation in (1) using Lactobacillus plantarum LP1406 and Lactobacillus rhamnosus F-B4-1 until the reducing sugar is exhausted, and then the solid-liquid is separated and the fermentation supernatant is collected, wherein the inoculation amount of Saccharomyces cerevisiae AMnb091, Lactobacillus plantarum LP1406, and Lactobacillus rhamnosus F-B4-1 is 3% of the volume of the raw material to be fermented; after fermentation, centrifugation is performed to obtain the supernatant; (3) Alcohol precipitation: The supernatant in (2) was concentrated by evaporation under reduced pressure, and anhydrous ethanol was added to the concentrate to a final ethanol concentration of 70%. The concentrate was allowed to stand at 4°C overnight, and the alcohol supernatant was collected by centrifugation, and concentrated to dryness by evaporation under reduced pressure to obtain an alcohol extract. (4) Redissolution: The dried alcohol extract was reconstituted with distilled water, and the pH was adjusted to 7 with sodium hydroxide solution. The precipitate was removed by centrifugation, and the supernatant was retained. (5) Purification: The supernatant in (4) was placed in a 500D dialysis bag, dialyzed with deionized water for 24 h, and then concentrated. DEAE Sephadex was added. TM In the A-25 chromatography column, pure water was used for elution, and the eluate was collected in 5 mL / tube at a flow rate of 1 mL / min. After high performance liquid chromatography detection, the oligosaccharide-rich components were collected, the eluate was combined and concentrated, and the concentrate was eluted by gel column chromatography Bio-Gel P-2, and the eluate was collected in segments.

[0023] The elutions with a peak at 30 min were collected by high performance liquid chromatography, combined and freeze-dried to obtain the seaweed oligosaccharide product. The seaweed oligosaccharide was analyzed by MS, and the molecular weight of the obtained seaweed oligosaccharide was 1031 Da. The purity of the obtained seaweed oligosaccharide was as high as 93.716%.

[0024] Liquid chromatography conditions: chromatographic column: Aminex HPX-87H, Bio-Rad, 300×7.8 mm, flow rate: 0.6 mL / min, mobile phase: 0.5 mM sulfuric acid solution, column temperature: 60°C, injection volume: 20 μL, detection wavelength: 210 nm.

[0025] MS conditions: High resolution mass spectrometer Bruker Q-TOF-MS (Impact , Germany). Mass spectrometry conditions: The mass spectrometer used an electrospray ion source, and the ion source parameters were as follows: capillary voltage was 3500 V in positive ion mode, 3000 V in negative ion mode, dry gas flow rate (DryGas) was 8 L / min; nebulizer pressure (Nebulizer) was 2.0 Bar, dry gas temperature (Drv Temp) was 220 °C, and the mass number detection range was 50-1500.

[0026] The HPLC chromatogram of the seaweed oligosaccharide prepared in this example is as follows: Figure 1 As shown in the attached figure, the ion flow diagram of N-acetylglucosamine mannopentaose (Man5GlcNAc-I) in the prepared seaweed oligosaccharide is shown in the attached figure. Figure 2 shown.

[0027] The structure of the above-mentioned seaweed oligosaccharide is as follows after mass spectrometry detection: .

[0028] Example 2 The difference from Example 1 is that during the re-dissolution process, the pH value was adjusted to 7.5, and the other steps were the same as Example 1. The purity of the final product was tested to reach 90.016%.

[0029] Example 3 The difference from Example 1 is that in the purification step, the flow rate is adjusted to 0.8 mL / min; the rest is the same as Example 1; and the purity of the final product is tested to be 93.02%.

[0030] Example 4 The difference from Example 1 is that in the purification step, the flow rate is adjusted to 1.1 mL / min; the rest is the same as Example 1; and the purity of the final product is tested to be 92.583%.

[0031] Example 5 The difference from Example 1 is that the dialysis time is 22 hours, and the rest is the same as Example 1; the purity of the final product is 92.152% after testing.

[0032] Example 6 The difference from Example 1 is that the dialysis time is 26 hours, and the rest is the same as Example 1; the purity of the final product is 93.463% after testing.

[0033] Example 7 The difference from Example 1 is that the kelp is crushed, and the other aspects are exactly the same as Example 1. After testing, the purity of the final product reaches 90.256%.

[0034] Example 8 The difference from Example 1 is that Sargassum fusiformis is crushed, and the rest is exactly the same as Example 1. After testing, the purity of the final product reaches 89.724%.

[0035] Example 9 The difference from Example 1 is that the Sargassum is crushed, and the other aspects are exactly the same as Example 1. After testing, the purity of the final product reaches 90.107%.

[0036] It can be seen from Examples 7-8 that the method of the present invention is not only applicable to the kelp in Example 1, but also applicable to other seaweed raw materials.

[0037] Comparative Example 1 The difference from Example 1 is that the purification method is different, specifically: when purifying seaweed oligosaccharides, the pH of the alcohol extract is not regulated, and the alcohol supernatant is directly subjected to column chromatography; the other steps are the same.

[0038] The HPLC chromatogram of the obtained seaweed oligosaccharide product is as follows: Figure 4 As shown, other impurities such as alkaloids in the final product are difficult to remove, and the purity of the target seaweed oligosaccharides obtained by HPLC is 76.199%.

[0039] Comparative Example 2 The difference from Example 1 is that the purification method is different, specifically: when purifying seaweed oligosaccharides, only DEAE Sephadex is used TM A-25 column chromatography was used instead of Bio-Gel P-2 gel column chromatography for purification; the other steps were the same.

[0040] The HPLC chromatogram of the obtained seaweed oligosaccharide product is as follows: Figure 5 As shown, it is obvious that other impurity oligosaccharides in the final product are difficult to remove. HPLC analysis shows that the purity of the target seaweed oligosaccharide is 51.246%.

[0041] Comparative Example 3 The difference from Example 1 is that only the gel column chromatography Bio-Gel P-2 is used for purification; the other steps are the same.

[0042] The HPLC chromatogram of the obtained seaweed oligosaccharide product is as follows: Figure 6 As shown in the figure, a large amount of organic acid impurities still remain in the obtained seaweed oligosaccharide product. High performance liquid chromatography analysis shows that the content of oligosaccharides in the final product is low, only 43.708%.

[0043] Comparative Example 4 The difference from Example 1 is that a DEAE cellulose DE-52 column is used instead of a DEAE Sephadex TMA-25 is subjected to chromatography, specifically: The supernatant was placed in a 500D dialysis bag, dialyzed with deionized water for 24 hours, then concentrated, added to a DEAE cellulose DE-52 chromatography column, eluted with pure water, and the eluate was collected. After the eluate was concentrated, it was eluted with a gel column chromatography Bio-Gel P-2, and the eluate was collected in segments. The other steps were the same.

[0044] The HPLC chromatogram of the obtained seaweed oligosaccharide product is as follows: Figure 7 As shown in the figure, some organic acid impurities still remain in the obtained seaweed oligosaccharide product. High performance liquid chromatography analysis shows that the content of oligosaccharides in the final product is low, at 59.462%.

[0045] The HPLC chromatograms of the comparative examples are shown in the attached figure. Figure 4-7 shown.

[0046] Test example The effects of the seaweed oligosaccharides prepared in each embodiment and comparative example and commercially available seaweed oligosaccharides on treating Parkinson's disease were evaluated using an in vitro Parkinson's disease evaluation method, as follows: SH-SY5Y neuroblastoma cells were cultured in a culture flask containing DMEM / F12 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics; the cells were cultured in a 5% carbon dioxide incubator at 37°C. The cells were seeded in a 96-well plate (12,000 cells / well) and incubated overnight to reach the desired confluence, and the cells were treated with 1 mm MPP+ (diluted with dimethyl sulfoxide) for 24 hours to establish a Parkinson's cell model.

[0047] Take the seaweed oligosaccharide prepared in Example 1, dilute it with culture medium, and the concentration is 50μg / mL, 200μg / mL and 1000μg / mL, and dilute the seaweed oligosaccharide prepared in Comparative Examples 1-4 and commercially available samples, so that the final concentration of these samples is 200μg / mL, place the cells in the culture medium containing different concentrations of each sample and let it stand for 12h, then add 20μL MTT (thiazolyl blue), after 4h, discard the culture medium, add 100μL DMSO (dimethyl sulfoxide), shake in the dark for 10min, and detect the cell survival rate. Take levodopa (5μM) as the positive control, detect the cell survival rate of each embodiment and comparative control group, and the results are shown in Table 1.

[0048] Table 1 Effects of seaweed oligosaccharides prepared by different processes on the survival rate of Parkinson's model cells Group Cell survival rate (%) Normal control group 100 PD model group 55.97 Positive control group 115.16 Example 1 (50 μg / mL) 86.11 Example 1 (200 μg / mL) 90.37 Example 1 (1000 μg / mL) 102 Comparative Example 1 76.62 Comparative Example 2 76.46 Comparative Example 3 75.66 Comparative Example 4 76.14 Commercially available samples 62.05 The data in the above table correspond to the Figure 3, the PD model group corresponds to the "model" in the figure, the positive control group corresponds to "levodopa", Example 1 (50 μg / mL) corresponds to seaweed oligosaccharides 50 μg / mL, Example 1 (200 μg / mL) corresponds to seaweed oligosaccharides 200 μg / mL, and Example 1 (1000 μg / mL) corresponds to seaweed oligosaccharides 1000 μg / mL.

[0049] In the above table, the effects of commercially available samples and seaweed oligosaccharides prepared by the comparative example process on the survival rate of Parkinson's model cells are compared. The cell survival rate of the sample of 50 μg / mL in Example 1 can reach about 86.11%; the cell survival rate of the sample of 1000 μg / mL can reach 102%. This shows that the seaweed oligosaccharide sample prepared in Example 1 can significantly increase the cell survival rate, and is positively correlated with the concentration of seaweed oligosaccharides, proving that the seaweed oligosaccharides of the present invention have a significant effect on improving the survival rate of Parkinson's model cells.

[0050] However, the products in comparative examples 1-4, due to the change of the purification step, were tested to have a cell survival rate of only about 76%, while at the same concentration, the cell survival rate of the product in Example 1 was about 90.37%, far exceeding comparative examples 1-4 and similar products on the market. This shows that purification is a key step, and if the seaweed oligosaccharide is not purified by appropriate means, the effect of the product will be compromised, but compared with ordinary products on the market, the products in the comparative examples of the present invention are also better than similar products on the market.

[0051] Therefore, it can be seen from the above table that the seaweed oligosaccharide prepared by the present invention has the effect of repairing nerve damage and has a good effect on the treatment of Parkinson's disease.

[0052] In view of the above experiments, it is proved that the seaweed oligosaccharides of the present invention have the above-mentioned effects and can be applied to any product of oral liquid, compressed candy, capsule, solid granule, and solid beverage. The preparation process of the above-mentioned products is relatively conventional, and the specific preparation process of the above-mentioned products will not be repeated here.

Claims

1. Application of seaweed oligosaccharides in the preparation of products for assisting the treatment of Parkinson's disease, characterized in that: The seaweed oligosaccharide has the following structure: 。 2. The use according to claim 1, characterized in that The seaweed oligosaccharide is obtained by the following preparation method: the cleaned and dried seaweed is crushed, enzymolyzed, fermented, the fermented supernatant is evaporated and concentrated, then alcohol extraction is performed, the alcohol supernatant is concentrated under reduced pressure, purified, and freeze-dried to obtain the seaweed oligosaccharide product.

3. The use according to claim 2, characterized in that The purification is as follows: The alcohol supernatant after vacuum concentration was dried, the extract was reconstituted with distilled water, the pH value was adjusted to 7-8, and the precipitate was removed by centrifugation; the supernatant was placed in a dialysis bag, dialyzed with deionized water, concentrated, and DEAE Sephadex was added. TM The A-25 chromatography column was eluted with pure water, and the eluate was collected. The eluate was concentrated and then eluted with the gel column Bio-Gel P-2, and the eluate was collected in sections.

4. The use according to claim 3, characterized in that The pH value is adjusted using sodium hydroxide solution, and the dialysis bag is 500D.

5. The use according to claim 3, characterized in that The solution was dialyzed against deionized water for 24 h and then concentrated.

6. The use according to claim 1, characterized in that The product for assisting the treatment of Parkinson's disease includes any one of oral liquid, compressed candy, capsule, solid granule, and solid beverage.

7. The use according to claim 2, characterized in that The seaweed is selected from any one of wakame, kelp, hijiki, sargassum and kelp.

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