1, 4-alpha-D-galacturonic acid oligosaccharide as well as preparation method and application thereof
Preparation of 1,4-α-D-galacturonic oligosaccharide by hydrolysis and purification of polygalacturonic pectin has solved the research problem of lack of such substances in the prior art, and achieved effective therapeutic effects on Alzheimer's disease and ischemic stroke.
Patent Information
- Application Number
- CN202510119735.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-25
- Publication Date
- 2025-07-25
AI Technical Summary
There is currently a lack of research on 1,4-α-D-galacturonate oligosaccharides, and the incidence and mortality of brain diseases such as Alzheimer's disease and ischemic stroke are increasing, and new preventive and therapeutic drugs are needed.
By hydrolyzing polygalacturonic pectin, using hydrochloric acid, sulfuric acid, trifluoroacetic acid, hydrobromic acid, hydroiodoic acid or perchloric acid as acid conditions, combined with gel permeation chromatography purification and lyophilization treatment, 1,4-α-D-galacturonic oligosaccharides are prepared for the preparation of health care products and drugs for the treatment of brain diseases.
The prepared 1,4-α-D-galacturonate oligosaccharides showed significant therapeutic effects on brain diseases such as Alzheimer's disease and ischemic stroke, which can significantly inhibit the apoptosis of nerve cells and cell damage under hypoxia and sugar deficiency conditions induced by Aβ25-35 oligomer, and have potential therapeutic potential.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine, and particularly relates to a 1,4-α-D-galacturonic acid oligosaccharide, a preparation method thereof, and uses thereof. Background Art
[0002] Pectin is widely distributed in the cell walls and middle lamellae of fruits and vegetables, such as citrus, apples, lemons, oranges, bananas, etc. Pectin is a structurally complex acidic heteropolysaccharide with a molecular weight ranging from several tens to tens of thousands of kDa. It has five different structural types, namely: homogalacturonoglycan (HG), rhamnogalacturonan-I (RG-I), rhamnogalacturonan-II (RG-II), xylogalacturonan (XG), and arabinogalactan (AG). Among them, homogalacturonoglycan (HG) accounts for more than 65%. However, there is currently no report on 1,4-α-D-galacturonic acid oligosaccharide.
[0003] In recent years, with the improvement of the public's living standards and the increasing aging of the population structure, the incidence and mortality of brain diseases are on the rise. Therefore, it is urgent to develop new drugs for the prevention and treatment of brain diseases (such as AD, ischemic stroke, etc.). Summary of the Invention
[0004] The object of the present invention is to address the above problems existing in the prior art and propose a 1,4-α-D-galacturonic acid oligosaccharide, a preparation method thereof, and uses thereof.
[0005] The first object of the present invention can be achieved by the following technical solutions:
[0006] A 1,4-α-D-galacturonic acid oligosaccharide, characterized in that its structural formula is as shown in (I):
[0007]
[0008] Wherein, each occurrence of M independently selects from hydrogen or a metal cation; n is one or more integers selected from 2 - 100;
[0009] Its composition comprises one or more 1,4-α-D-galacturonic acid oligosaccharides having the structure represented by formula (I).
[0010] In the above 1,4-α-D-galacturonic acid oligosaccharide, the cation comprises one or more selected from the following: lithium ion, sodium ion, potassium ion, beryllium ion, magnesium ion, calcium ion, iron ion, ferrous ion, zinc ion, selenium ion, vanadium ion, tin ion, and strontium ion.
[0011] In the above-mentioned 1,4-α-D-galacturonic acid oligosaccharides, n is one or more integers selected from 2 to 100.
[0012] The second object of the present invention can be achieved by the following technical solutions:
[0013] A method for preparing 1,4-α-D-galacturonic acid oligosaccharides, characterized by comprising the following steps:
[0014] (a) Hydrolysis of polygalacturonic acid (HG-type pectin); (b) Purification, concentration, and lyophilization of the hydrolysis solution.
[0015] In the above-mentioned method for preparing 1,4-α-D-galacturonic acid oligosaccharides, the hydrolysis in step (a) is carried out in the presence of an acid; the purification in step (b) is carried out using a gel permeation chromatography packing material.
[0016] In the above-mentioned method for preparing 1,4-α-D-galacturonic acid oligosaccharides, the acid is one or more of hydrochloric acid, sulfuric acid, trifluoroacetic acid, hydrobromic acid, hydroiodic acid, and perchloric acid.
[0017] The third object of the present invention can be achieved by the following technical solutions:
[0018] An application of 1,4-α-D-galacturonic acid oligosaccharides, characterized in that a composition is formed by one or more 1,4-α-D-galacturonic acid oligosaccharides having the structure represented by formula (I), and is applied to health products and the preparation of drugs for preventing and treating brain diseases.
[0019] In the above-mentioned application of 1,4-α-D-galacturonic acid oligosaccharides, the brain diseases are selected from Alzheimer's disease, transient ischemic attack, stroke, ischemic stroke, cerebral infarction, moyamoya disease, or vascular dementia.
[0020] Compared with the prior art, the present invention has the following advantages:
[0021] The 1,4-α-D-galacturonic acid oligosaccharides of the present invention are products obtained by hydrolyzing, gel-purifying, concentrating, and lyophilizing HG-type pectin (polygalacturonic acid). The 1,4-α-D-galacturonic acid oligosaccharides of the present invention contain 2-20 α-D-galacturonic acid oligosaccharide repeating units prepared by the preparation method. The 1,4-α-D-galacturonic acid oligosaccharides of the present invention have a significant effect on treating encephalopathies such as Alzheimer's disease and ischemic stroke. Description of the Drawings
[0022] Figure 1 is the 1H NMR spectrum of polygalacturonic acid;
[0023] Figure 2 It is the chromatogram of the oligosaccharide solution after hydrolysis;
[0024] Figure 3 It is the chromatogram of the purified and concentrated oligosaccharide solution;
[0025] Figure 4 It is the mass spectrum of 1,4-α-D-galacturonic acid oligosaccharide disaccharide (n = 2 in formula I);
[0026] Figure 5 It is the mass spectrum of 1,4-α-D-galacturonic acid oligosaccharide trisaccharide (n = 3 in formula I);
[0027] Figure 6 It is the mass spectrum of 1,4-α-D-galacturonic acid oligosaccharide tetrasaccharide (n = 4 in formula I);
[0028] Figure 7 It is the mass spectrum of 1,4-α-D-galacturonic acid oligosaccharide pentasaccharide (n = 5 in formula I);
[0029] Figure 8 It is the mass spectrum of 1,4-α-D-galacturonic acid oligosaccharide hexasaccharide (n = 6 in formula I);
[0030] Figure 9 It is the mass spectrum of 1,4-α-D-galacturonic acid oligosaccharide heptasaccharide (n = 7 in formula I);
[0031] Figure 10 It is the mass spectrum of 1,4-α-D-galacturonic acid oligosaccharide octasaccharide (n = 8 in formula I);
[0032] Figure 11 It is the mass spectrum of 1,4-α-D-galacturonic acid oligosaccharide nonasaccharide (n = 9 in formula I);
[0033] Figure 12 It is the mass spectrum of 1,4-α-D-galacturonic acid oligosaccharide decasaccharide (n = 10 in formula I);
[0034] Figure 13 It is the graph of the effect of 1,4-α-D-galacturonic acid oligosaccharide composition on the survival rate of normally cultured SH-SY5Y cells;
[0035] Figure 14 It is the graph of the effect of 1,4-α-D-galacturonic acid oligosaccharide composition on the survival rate of Aβ25-35-induced apoptotic SH-SY5Y cells;
[0036] Figure 15 It is the graph of the effect of 1,4-α-D-galacturonic acid oligosaccharide composition on the survival rate of normally cultured SH-SY5Y cells;
[0037] Figure 16Effect of 1,4-α-D-galacturonic acid oligosaccharide composition on the survival rate of OGD-induced SH-SY5Y cells Detailed implementation mode
[0038] The following are specific embodiments of the present invention and in combination with the accompanying drawings, the technical solutions of the present invention will be further described, but the present invention is not limited to these embodiments.
[0039] Example 1
[0040] A 1,4-α-D-galacturonic acid oligosaccharide, the structural formula of which is shown in (I):
[0041]
[0042] Wherein, each occurrence of M is independently selected from hydrogen or a metal cation; n is one or more integers selected from 2-100.
[0043] Wherein, the cation comprises one or more selected from the following: lithium ion, sodium ion, potassium ion, beryllium ion, magnesium ion, calcium ion, iron ion, ferrous ion, zinc ion, selenium ion, vanadium ion, tin ion and strontium ion. Most preferably, n is one or more integers selected from 2, 3, 4, 5, 6, 7, 8, 9 and 10.
[0044] Wherein, the composition thereof is formed by one or more 1,4-α-D-galacturonic acid oligosaccharides having the structure represented by formula (I).
[0045] Example 2
[0046] A preparation method of 1,4-α-D-galacturonic acid oligosaccharide, the specific steps are as follows:
[0047] (1) Weigh 500 mg of polygalacturonic acid and add it into the degradation bottle, and measure 100 ml of 1 M trifluoroacetic acid aqueous solution and add it thereto, and stir well until a homogeneous polygalacturonic acid suspension is obtained.
[0048] (2) Place the degradation bottle in an oven at 100 °C under stirring conditions and degrade for 2 hours.
[0049] (3) Take out the degradation solution, let it cool, and adjust the pH to neutral with sodium hydroxide solution.
[0050] (4) Purify the neutralized solution by G25 gel permeation chromatography, prepare different oligosaccharide components, concentrate and freeze-dry to obtain a powdery solid. The yields of representative oligosaccharide components are shown in the following table, and the yield is the ratio of the mass of the obtained oligosaccharide powder to the mass of the substrate.
[0051] Oligosaccharide component Yield % Trisaccharide 3.6 Tetrasaccharide 2.9 Pentasaccharide 2.5 Hexasaccharide 2.3 Heptasaccharide 2.1 Oligosaccharide composition (2 - 10 saccharides) 20.1
[0052] Table 1, Total Yield after Hydrolysis and Purification of Representative Oligosaccharide Components
[0053] Confirmation of the Relative Configuration and Linkage Mode of 1,4-α-D-Galacturonic Acid Oligosaccharides
[0054] (1) Analytical Method
[0055] Accurately weigh 30 mg of polygalacturonic acid used in step (1) of Example 1 and dissolve it in 0.6 ml of heavy water (99.9% atom D). Perform 1H spectrum acquisition using a Bruker 400 MHz nuclear magnetic resonance spectrometer. The 1H spectrum acquisition conditions are as follows: the number of scans for the hydrogen spectrum is 16 times, and the test temperature is room temperature.
[0056] (2) Results
[0057] The 1H NMR spectrum of polygalacturonic acid is as Figure 1 shown. It can be seen from the figure that there is a characteristic peak at a chemical shift of 5.4 ppm, which is the anomeric H signal of α-galacturonic acid linked in the 1→4 manner. Combining with the fact that the absolute configuration of pyranose in nature is all D-type. Therefore, the fine structure of the polygalacturonic acid we used is 1,4-α-D-galacturonic acid glycan, and the oligosaccharides obtained after acid hydrolysis are 1,4-α-D-galacturonic acid oligosaccharides.
[0058] Confirmation of the Fine Structure of 1,4-α-D-Galacturonic Acid Oligosaccharides
[0059] (1) Analytical Method
[0060] Take appropriate amounts of the hydrolyzed oligosaccharide solution and the purified and concentrated oligosaccharide solution respectively for size exclusion ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry (SEC-UPLC-MS) (QExactive Plus, Thermo Fisher Scientific, USA) analysis. The chromatographic conditions are as follows: ZenixSEC80 size exclusion chromatography column (7.8×300 mm, Sepax); detection wavelength: 214 nm; mobile phase: A is 50 mM ammonium acetate aqueous solution, B is methanol, isocratic elution with 80% A for 20 min; flow rate: 1 ml / min. The mass spectrometry conditions are as follows: negative ion mode; scanning range (m / z): 150 - 2250.
[0061] (2) Results
[0062] The chromatograms of the hydrolyzed oligosaccharide solution and the purified and concentrated oligosaccharide solution are respectively as Figure 2 and Figure 3 shown. The mass spectra and analytical assignments of 1,4-α-D-galacturonic acid oligosaccharides from disaccharide to decasaccharide are respectively as Figures 4 - 12 shown.
[0063] Example 3
[0064] Use of 1,4-α-D-galacturonic acid oligosaccharides, a composition formed by one or more 1,4-α-D-galacturonic acid oligosaccharides having the structure represented by formula (I), is applied to health products and the preparation of drugs for preventing and treating brain diseases.
[0065] In the above application of 1,4-α-D-galacturonic acid oligosaccharides, the brain diseases are selected from Alzheimer's disease, transient ischemic attack, stroke, ischemic stroke, cerebral infarction, moyamoya disease, or vascular dementia.
[0066] Apoptosis inhibitory effect of 1,4-α-D-galacturonic acid oligosaccharide composition on Aβ oligomer-induced human neuroblastoma cells
[0067] (1) Method
[0068] Human neuroblastoma cells SH-SY5Y (Cell Bank of the Chinese Academy of Sciences) in the logarithmic growth phase were digested with trypsin to prepare a cell suspension, and added to a 96-well plate at an appropriate cell concentration of 100 μL / well, with 6 replicates, and cultured in a 37°C 5% CO2 cell incubator for 16 h. The supernatant was aspirated, and at the same time, SH-SY5Y cells were given Aβ25-35 and 1,4-α-D-galacturonic acid oligosaccharide composition. After 24 h of culture, 20 μL / well of 5 mg / mL MTT solution was added, and the culture was continued for 4 h. All the supernatant was discarded, 150 μL / well of DMSO was added, and it was oscillated on a micro oscillator for 5 min to completely dissolve the crystals. The absorbance value was measured at wavelengths of 570 nm and 630 nm with a full-wavelength microplate reader, and the cell survival rate was calculated.
[0069] (2) Results
[0070] The effects of 1,4-α-D-galacturonic acid oligosaccharide composition on the survival rates of normally cultured SH-SY5Y and Aβ25-35-induced apoptotic cells are as Figure 13 and Figure 14 shown. It can be seen from Figure 13 that there is no significant difference in the cell survival rates of the low-dose group, medium-dose group, and high-dose group compared with the normal group. This indicates that the 1,4-α-D-galacturonic acid oligosaccharide composition has no toxicity and proliferative activity on SH-SY5Y cells.
[0071] It can be seen from Figure 14It can be seen that the survival rate of SH-SY5Y cells in the Aβ25-35 oligomer induction group was significantly lower than that in the normal group (p<0.001). This indicates that Aβ25-35 oligomers significantly inhibit cell survival, suggesting successful model establishment. The cell survival rates in the low-dose group, medium-dose group, and high-dose group were higher than those in the model group, and there were significant differences between the medium- and high-dose groups and the model group (p<0.05), showing a good dose-dependent relationship. This indicates that the 1,4-α-D-galacturonic acid oligosaccharide composition has a depolymerization or binding effect on aβ oligomers, thereby inhibiting the apoptosis of human neuroblastoma cells induced by them, and has the potential to treat Alzheimer's disease.
[0072] Inhibitory effect of 1,4-α-D-galacturonic acid oligosaccharide composition on apoptosis of human neuroblastoma cell SH-SY5Y induced by oxygen and glucose deprivation (OGD) model (1) Method
[0073] In the OGD model, the nutrient supply of cells is deprived, so this model can be used to study the protective effect of drugs on cells under hypoxic and hypoglycemic conditions.
[0074] Culture under normal conditions: Take HT-22 cells cultured under normal conditions, adjust the cell number to 2×104 cells / ml; inoculate the cells on a 96-well culture plate at 100 μl / well. The cells were divided into a low-dose group, a medium-dose group, a high-dose group, and a normal group; each group had 4 parallel groups (n = 4). The cells were pre-cultured with high-glucose culture medium for 12 hours. Add 10 μl of the 1,4-α-D-galacturonic acid oligosaccharide composition obtained in Example 1 with concentrations of 12.5 μg / ml, 25 μg / ml, and 50 μg / ml prepared with PBS to the low-dose group, medium-dose group, and high-dose group respectively, and add the same volume of PBS to the normal group. Then, place the culture plate in a constant temperature incubator at 5% CO2 and 37 °C for 12 hours. Measure the cell survival rate.
[0075] Culture under oxygen-glucose deprivation conditions: Take the normally cultured HT-22 cells, adjust the cell number to 2×104 cells / ml; inoculate the cells on a 96-well culture plate at 100 μl / well. Divide the cells into a low-dose group, a medium-dose group, a high-dose group, and an OGD group; each group has 4 parallel groups (n = 4). Pre-culture the cells with high-glucose culture medium for 12 hours. Then, aspirate the high-glucose culture medium, wash the cells with sugar-free DMEM culture medium, and finally replace the high-glucose culture medium with DMEM culture medium. Add 10 μl of the 1,4-α-D-galacturonic acid oligosaccharide composition obtained in Example 1 with concentrations of 12.5 μg / ml, 25 μg / ml, and 50 μg / ml prepared with PBS to the low-dose group, medium-dose group, and high-dose group respectively. Place the culture plate in an oxygen-deprivation chamber (95% N2, 5% CO2), and culture at a constant temperature of 37°C for 12 hours. Measure the cell viability.
[0076] (2) Results
[0077] The effects of the 1,4-α-D-galacturonic acid oligosaccharide composition on the cell viability of normally cultured SH-SY5Y and OGD-induced apoptotic cells are as Figure 15 and Figure 16 shown. It can be seen from Figure 15 that there is no significant difference in the cell viability of the low-dose group, medium-dose group, and high-dose group compared with the normal group. This indicates that the 1,4-α-D-galacturonic acid oligosaccharide composition has no toxicity and proliferative activity on SH-SY5Y cells.
[0078] It can be seen from Figure 16 that the cell viability of SH-SY5Y cells in the OGD-induced group is significantly decreased compared with the normal group (p < 0.001). This indicates that OGD significantly inhibits cell survival, suggesting that the model is successfully established. The cell viability of the low-dose group, medium-dose group, and high-dose group is increased compared with the model group, and there is a significant difference between the medium-dose and high-dose groups and the model group (p < 0.05), showing a good dose-dependent relationship. This indicates that the 1,4-α-D-galacturonic acid oligosaccharide composition has an inhibitory effect on the apoptosis of OGD-induced human neuroblastoma cells and has the potential to treat ischemic stroke.
[0079] As described above, it is only the preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.
Claims
1. A 1,4-α-D-galacturonic acid oligosaccharide, characterized in that, As shown in its structural formula (I): Wherein, each occurrence of M is independently selected from hydrogen or a metal cation; n is one or more integers selected from 2 - 100.
2. The 1,4-α-D-galacturonic acid oligosaccharide according to claim 1, characterized in that, The cation comprises one or more selected from the following: lithium ion, sodium ion, potassium ion, beryllium ion, magnesium ion, calcium ion, iron ion, ferrous ion, zinc ion, selenium ion, vanadium ion, tin ion, and strontium ion.
3. The 1,4-α-D-galacturonic acid oligosaccharide according to claim 1, characterized in that, The n is one or more integers selected from 2 - 100.
4. A method for preparing 1,4-α-D-galacturonic acid oligosaccharide according to claim 1, characterized in that, Comprising the following steps: (1) Under optionally stirring conditions, add polygalacturonic acid to an aqueous hydrochloric acid solvent to obtain a suspension containing polygalacturonic acid; (2) React the above suspension of polygalacturonic acid in a high-temperature environment for 2 - 10 hours under optionally stirring conditions; (3) After acidolysis, adjust the pH of the solution to neutral with a sodium hydroxide solution; (4) Purify the above neutral solution with a G25 type gel permeation chromatography packing material; (5) The purified solution obtained is concentrated under optionally conditions, adjusted to neutral pH, and freeze-dried.
5. The preparation method of 1,4-α-D-galacturonic acid oligosaccharide according to claim 4, characterized in that, In step (1), the mass-to-volume ratio of the lacturonic acid to the acidic solvent is preferably 1:100 - 500 g / mL; the acid includes but is not limited to: hydrochloric acid, sulfuric acid, sulfurous acid, phosphoric acid, metaphosphoric acid, phosphorous acid, hypophosphorous acid, pyrophosphoric acid, polyphosphoric acid, hydrobromic acid, hydroiodic acid, perchloric acid; the concentration of the acidic solvent is preferably 100 mM - 3 M.
6. The preparation method of 1,4-α-D-galacturonic acid oligosaccharide according to claim 4, characterized in that, In step (2), the high-temperature environment is preferably 80 - 150 °C; the reaction time is preferably 2 - 10 hours.
7. The preparation method of 1,4-α-D-galacturonic acid oligosaccharide according to claim 4, characterized in that, In step (4), it is preferably to use gel permeation chromatography for purification, and most preferably a G25 type gel permeation chromatography packing material.
8. The preparation method of 1,4-α-D-galacturonic acid oligosaccharide according to claim 4, characterized in that, In step (5), an alkali metal salt or an alkaline earth metal salt is used to adjust the pH value to neutral.
9. Use of the 1,4-α-D-galacturonic acid oligosaccharide according to claim 1, characterized in that, A composition is formed by one or more 1,4-α-D-galacturonic acid oligosaccharides having the structure represented by formula (I), and is applied to health products and the preparation of drugs for preventing and treating brain diseases.
10. Use of the 1,4-α-D-galacturonic acid oligosaccharide according to claim 9, characterized in that, The brain diseases are selected from Alzheimer's disease, transient ischemic attack, stroke, ischemic stroke, cerebral infarction, moyamoya disease, or vascular dementia.