Application of seaweed oligosaccharides in the preparation of products for the auxiliary treatment of Parkinson's disease

Abstract

The present invention belongs to the field of application of seaweed products, and specifically relates to the application of seaweed oligosaccharides in the preparation of products for the auxiliary treatment of Parkinson's disease. The present invention uses probiotics to biotransform seaweed oligosaccharide components, and obtains seaweed oligosaccharides with the auxiliary treatment of Parkinson's disease through pH control, ion exchange chromatography, and gel chromatography separation and purification. The greatest innovation of this method is the use of pH control to crudely purify the fermentation liquid after ethanol extraction, and DEAE Sephadex TM The extract was purified by A-25 column chromatography and Bio-Gel P-2 gel column chromatography, increasing the purity of the obtained seaweed oligosaccharides to approximately 93%. The seaweed oligosaccharides obtained by this method have a beneficial effect on the treatment of Parkinson's disease, providing a new and safe product for the adjunctive treatment of Parkinson's disease.

Description

Technical Field

[0001] The present invention belongs to the application field of seaweed functional products, and in particular relates to the application of seaweed oligosaccharide in the preparation of 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 easier to be absorbed and utilized by organisms.

[0003] Parkinson's disease is a neurodegenerative disorder caused by the selective loss of dopaminergic neurons. It is predominantly seen in middle-aged and elderly people, and its incidence is increasing annually with aging. Currently, there are no effective treatments for Parkinson's disease, and most existing drugs can only temporarily alleviate symptoms but cannot prevent or slow the progression of the disease.

[0004] Currently, research on seaweed oligosaccharides is mostly focused on the preparation methods 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 between the structure and function of seaweed oligosaccharides. There is even less literature on the mechanism of the relationship 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 disease auxiliary treatment products. Seaweed oligosaccharide components are obtained through probiotic biotransformation, and seaweed oligosaccharides with a specific structure are obtained through separation and purification. The seaweed oligosaccharides with this structure can be used to prepare Parkinson's disease auxiliary treatment products, providing a new, effective and safe product for the auxiliary treatment of Parkinson's disease.

[0006] The technical solutions of the present invention are as follows:

[0007] The present invention provides an application of seaweed oligosaccharide in preparing a product for assisting in the treatment of Parkinson's disease. The seaweed oligosaccharide has the following structure:

[0008] .

[0009] Preferably, seaweed oligosaccharides are obtained by the following preparation method: the cleaned and dried seaweed is crushed, enzymatically hydrolyzed, fermented, the fermented supernatant is evaporated and concentrated, and then subjected to alcohol extraction, the alcohol supernatant is concentrated under reduced pressure, purified, and freeze-dried to obtain the seaweed oligosaccharide product.

[0010] The seaweed is selected from any one of wakame, kelp, hijiki, sargassum and konbu.

[0011] 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.

[0012] 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.

[0013] 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%.

[0014] Preferably, the purification steps are as follows:

[0015] The alcohol supernatant was concentrated under reduced pressure and dried. The extract was redissolved 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 and dialyzed with deionized water for 24 hours before being concentrated. DEAE Sephadex was added. TM The mixture was eluted with pure water in an A-25 chromatography column, and the eluate was collected. The eluate was concentrated and then eluted in a gel column chromatography Bio-Gel P-2, and the eluate was collected in sections.

[0016] In the present invention, after the seaweed is subjected to enzymatic fermentation, the supernatant obtained by fermentation is eluted with ethanol, which is beneficial for dissolving effective ingredients such as oligosaccharides in the alcohol supernatant, removing impurities such as large molecular proteins and polysaccharides, and facilitating further separation and purification.

[0017] Preferably, the Parkinson's disease auxiliary treatment product includes any one of oral liquid, compressed candy, capsule, solid granule, and solid beverage.

[0018] Preferably, the molecular weight of seaweed oligosaccharide is 1031 Da.

[0019] The beneficial effects of the present invention are:

[0020] (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;

[0021] (2) The present invention obtains seaweed oligosaccharide components through probiotic biotransformation, and obtains seaweed oligosaccharides with a specific structure through separation and purification. 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

[0022] Figure 1 This is the HPLC peak diagram of the seaweed oligosaccharide prepared in Example 1 of the present invention;

[0023] Figure 2 This is the ion current diagram of N-acetylglucosamine mannopentaose (Man5GlcNAc-I) in the seaweed oligosaccharide prepared in Example 1 of the present invention;

[0024] Figure 3 The effects of different treatments of the present invention on cell survival in a Parkinson's cell model;

[0025] Figure 4 This is a high performance liquid chromatogram of seaweed oligosaccharides obtained by the method in Comparative Example 1 of the present invention;

[0026] 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;

[0027] 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;

[0028] 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

[0029] In order to enable those skilled in the art to better understand the present invention, the present invention will be further explained in conjunction with specific embodiments.

[0030] In the following examples, 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.

[0031] Lactobacillus plantarum was deposited in the China Center for Type Culture Collection on November 29, 2021, address: Wuhan University, Wuhan, China, with the deposit number: CCTCC NO: M 20211500, and the classification name: Lactobacillus plantarum LP1406 Lactiplantibacillus plantarum LP1406.

[0032] 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.

[0033] Example 1

[0034] The preparation method of seaweed oligosaccharides comprises the following steps:

[0035] (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 enzymatic hydrolysis conditions were: enzymatic hydrolysis at 55°C for 2 h; after enzymatic hydrolysis, the enzyme was inactivated at 95°C for 10 min.

[0036] (2) Fermentation: aerobic fermentation was performed using Saccharomyces cerevisiae AMnb091, and then anaerobically fermented the enzymatic hydrolysate after enzyme inactivation in (1) using Lactobacillus plantarum LP1406 and Lactobacillus rhamnosus F-B4-1 until the reducing sugar was exhausted, solid-liquid separation was performed, and the fermentation supernatant was collected, wherein the inoculation amount of Saccharomyces cerevisiae AMnb091, Lactobacillus plantarum LP1406, and Lactobacillus rhamnosus F-B4-1 was 3% of the volume of the raw material to be fermented; after fermentation, the supernatant was obtained by centrifugation;

[0037] (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;

[0038] (4) Redissolution: The dried alcohol extract was re-dissolved in distilled water, and the pH was adjusted to 7 by adding sodium hydroxide solution. The precipitate was removed by centrifugation, and the supernatant was retained.

[0039] (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. TMIn 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 eluates were combined and concentrated, and the concentrate was eluted by gel column chromatography Bio-Gel P-2, and the eluate was collected in segments.

[0040] High performance liquid chromatography was used for determination, and the eluate with a peak at 30 min was collected, combined and freeze-dried to obtain the seaweed oligosaccharide product. MS analysis showed that the molecular weight of the obtained seaweed oligosaccharide was 1031 Da, and the purity of the obtained seaweed oligosaccharide was as high as 93.716%.

[0041] 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.

[0042] MS conditions: High resolution mass spectrometry Bruker Q-TOF-MS (Impact , Germany). Mass spectrometry conditions: The mass spectrometer used an electrospray ionization source with the following ion source parameters: capillary voltage 3500 V in positive ion mode, 3000 V in negative ion mode, dry gas flow rate (DryGas) of 8 L / min, nebulizer pressure (Nebulizer) of 2.0 Bar, and dry gas temperature (DrvTemp) of 220°C. The mass detection range was 50–1500.

[0043] The HPLC chromatogram of the seaweed oligosaccharide prepared in this example is as follows: Figure 1 As shown in the attached figure, the ion current of N-acetylglucosamine mannopentaose (Man5GlcNAc-I) in the prepared seaweed oligosaccharide is shown in the attached figure. Figure 2 shown.

[0044] The structure of the above-mentioned seaweed oligosaccharide is as follows after mass spectrometry detection:

[0045] .

[0046] Example 2

[0047] The difference from Example 1 is that during the re-dissolution process, the pH value was adjusted to 7.5. The other steps were the same as in Example 1. The purity of the final product was tested to reach 90.016%.

[0048] Example 3

[0049] 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 reaches 93.02% after testing.

[0050] Example 4

[0051] 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 reaches 92.583% after testing.

[0052] Example 5

[0053] The difference from Example 1 is that the dialysis time is 22 hours, and the other aspects are the same as Example 1; the purity of the final product reaches 92.152% after testing.

[0054] Example 6

[0055] The difference from Example 1 is that the dialysis time is 26 hours, and the other aspects are the same as Example 1; the purity of the final product reaches 93.463% after testing.

[0056] Example 7

[0057] 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%.

[0058] Example 8

[0059] The difference from Example 1 is that the sea lettuce is crushed, and the other aspects are exactly the same as Example 1. After testing, the purity of the final product reaches 89.724%.

[0060] Example 9

[0061] 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%.

[0062] 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.

[0063] Comparative Example 1

[0064] 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.

[0065] 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 oligosaccharide obtained by HPLC analysis is 76.199%.

[0066] Comparative Example 2

[0067] 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.

[0068] The HPLC chromatogram of the obtained seaweed oligosaccharide product is as follows: Figure 5 As shown in the figure, it is obvious that other impurity oligosaccharides in the final product are difficult to remove. The purity of the target seaweed oligosaccharide obtained by HPLC analysis is 51.246%.

[0069] Comparative Example 3

[0070] The difference from Example 1 is that only Bio-Gel P-2 gel column chromatography is used for purification; the other steps are the same.

[0071] 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%.

[0072] Comparative Example 4

[0073] The difference from Example 1 is that a DEAE cellulose DE-52 column was used instead of a DEAE Sephadex column. TM A-25 is subjected to chromatography, specifically:

[0074] The supernatant was placed in a 500D dialysis bag, dialyzed with deionized water for 24 hours, and then concentrated. The supernatant was added to a DEAE cellulose DE-52 chromatography column, eluted with pure water, and the eluate was collected. The eluate was concentrated and then eluted with a gel column chromatography Bio-Gel P-2. The eluate was collected in sections. The other steps were the same.

[0075] 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%.

[0076] The HPLC chromatograms of the comparative examples are shown in the attached figure. Figure 4-7 shown.

[0077] Test example

[0078] The effects of the seaweed oligosaccharides prepared in each example 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:

[0079] SH-SY5Y neuroblastoma cells were cultured in flasks containing DMEM / F12 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics; the cells were cultured in a 5% CO2 incubator at 37°C. The cells were seeded in 96-well plates (12,000 cells / well) and incubated overnight to reach the desired confluence. The cells were then treated with 1 mM MPP+ (diluted with dimethyl sulfoxide) for 24 hours to establish a Parkinson's disease cell model.

[0080] The seaweed oligosaccharides prepared in Example 1 were diluted with culture medium to concentrations of 50 μg / mL, 200 μg / mL, and 1000 μg / mL, respectively. The seaweed oligosaccharides prepared in Comparative Examples 1-4 and commercially available samples were diluted to a final concentration of 200 μg / mL. The cells were placed in culture medium containing different concentrations of each sample and allowed to stand for 12 hours. 20 μL of MTT (thiazolyl blue) was then added. After 4 hours, the culture medium was discarded and 100 μL of DMSO (dimethyl sulfoxide) was added. The cells were shaken in the dark for 10 minutes and the cell viability was detected. L-dopa (5 μM) was used as a positive control to detect the cell viability of the control group of each example and the comparative example. The results are shown in Table 1.

[0081] Table 1 Effects of seaweed oligosaccharides prepared by different processes on the survival rate of Parkinson's model cells

[0082] 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

[0083] 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 50 μg / mL of seaweed oligosaccharides, Example 1 (200 μg / mL) corresponds to 200 μg / mL of seaweed oligosaccharides, and Example 1 (1000 μg / mL) corresponds to 1000 μg / mL of seaweed oligosaccharides.

[0084] The table above compares the effects of commercially available samples and seaweed oligosaccharides prepared using the comparative example process on the survival rate of Parkinson's model cells. The 50 μg / mL sample in Example 1 achieved a cell survival rate of approximately 86.11%, while the 1000 μg / mL sample achieved a cell survival rate of 102%. This indicates that the seaweed oligosaccharide sample prepared in Example 1 can significantly increase cell survival rate, and is positively correlated with the concentration of the seaweed oligosaccharide, demonstrating that the seaweed oligosaccharide of the present invention has a significant effect on improving the survival rate of Parkinson's model cells.

[0085] However, due to the changes in the purification steps, the products in Comparative Examples 1-4 had a cell viability of only about 76%. At the same concentration, the cell viability of the product in Example 1 reached about 90.37%, far exceeding that of Comparative Examples 1-4 and similar commercial products. This shows that purification is a critical step. If the seaweed oligosaccharides are not purified by appropriate means, the product's effectiveness will be compromised. However, compared with ordinary commercial products, the products in the comparative examples of the present invention are still superior to similar commercial products.

[0086] 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.

[0087] 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 products is relatively conventional, and the specific preparation process of the above products will not be repeated here.

Claims

1. The application of seaweed oligosaccharide in the preparation of drugs for auxiliary 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, and 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 redissolved in 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, and concentrated, and DEAE Sephadex was added. TM The mixture was eluted with pure water in an A-25 chromatography column, and the eluate was collected. The eluate was concentrated and then eluted in a gel column chromatography Bio-Gel P-2, and the eluate was collected in sections.

4. The use according to claim 3, characterized in that Sodium hydroxide solution was used to adjust the pH value, and the dialysis bag was 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, wherein The drug for assisting in the treatment of Parkinson's disease includes any one of oral liquid, capsule, and solid granule.

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

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