Acetylated hawthorn polysaccharide, and preparation method and application thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to acetylated hawthorn polysaccharide as well as a preparation method and application thereof. The preparation method of the acetylated hawthorn polysaccharide comprises the following steps: homogenizing fresh hawthorn fruits in a low eutectic solvent, and extracting by a physical method to obtain an extract; performing alcohol precipitation and centrifugation on the extract to obtain crude polysaccharide; resolubilizing the crude polysaccharide in water, and performing deproteinization, concentration, dialysis and freeze-drying to obtain the acetylated hawthorn polysaccharide. The physical method is used in cooperation with the low eutectic solvent for extraction, which is green and pollution-free, can realize efficient preparation and effective separation of the acetylated hawthorn polysaccharide, and has a high acetylation degree. The acetylated hawthorn polysaccharide has an in-vitro and in-vivo regulating activity on intestinal mitochondrial homeostasis, has no toxic and side effects, is suitable for intestinal mitochondrial related disease groups, and is suitable for intestinal disease prevention of normal people.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to acetylated hawthorn polysaccharide as well as a preparation method and application thereof. BACKGROUND

[0002] Mitochondria, a double-membrane organelle existing in most cells, is the structure for energy production in cells, and can maintain the energy homeostasis and normal function of cells, especially high-energy cells. Intestinal epithelial cells have a high dependence on mitochondrial energy supply due to their rapid self-renewal rate and the functions of barrier, absorption and immune regulation. The health status of intestinal mitochondria directly determines the integrity of intestinal function. Studies have shown that the decline of intestinal mitochondrial function is an important cause of intestinal barrier damage, local chronic inflammation and microbial imbalance, and may further lead to systemic health problems. Therefore, effective intervention and maintenance of intestinal mitochondrial function are crucial for maintaining energy homeostasis and health of the body.

[0003] Diet is one of the most direct external factors for regulating mitochondrial function in the body. The intestine, as the main site for the digestion and absorption of nutrients, is crucial for maintaining intestinal homeostasis. However, the molecular mechanisms of how specific dietary nutrition strategies affect intestinal mitochondrial function to improve intestinal health are still unclear, which seriously hinders the development of effective nutritional intervention programs targeting intestinal mitochondrial targets. A variety of food-derived polysaccharides from fruits and vegetables, mushrooms, seaweed and Chinese herbal medicines have been confirmed to maintain intestinal health by regulating mitochondrial function, and the mechanisms involve regulating mitochondrial dynamics, autophagy and repairing intestinal barrier. However, the resources of polysaccharides with such activities are still relatively limited, and there are significant differences in the structure-activity relationship and efficacy of polysaccharides from different sources. Most of the studies are still limited to the preliminary observation of naturally extracted polysaccharides, and lack of exploration of targeted optimization or high-efficiency compounding strategies for specific structures, resulting in bottlenecks in the effectiveness of existing intervention programs and difficulty in prediction.

[0004] Acetylated polysaccharides are products of natural polysaccharides after chemical modification, which change the physicochemical properties of polysaccharides by introducing acetyl groups, such as changing the spatial conformation of polysaccharides, enhancing their binding capacity with specific bacterial surface glycoside hydrolases, and improving the degradation efficiency of polysaccharides by bacteria. Bacteria use degradation products as carbon sources or energy substrates to promote their own proliferation. However, there are few reports on the regulation of intestinal mitochondrial homeostasis by acetylated polysaccharides, and the underlying mechanisms are not clear. At the same time, the existing preparation of acetylated polysaccharides mainly uses chemical methods (such as acetic anhydride method), but the acetylated polysaccharides prepared by this method have problems such as easy destruction of structure, low purity and poor uniformity of product, and great environmental pressure. SUMMARY

[0005] The application aims to provide acetylated hawthorn polysaccharide and a preparation method and application thereof, so as to overcome the shortcomings of the prior art, realize efficient preparation and effective separation of the acetylated hawthorn polysaccharide by using a physical method and a low eutectic solvent, and realize high acetylation degree, in-vitro and in-vivo regulation of intestinal mitochondrial homeostatic activity, and no toxic side effects, which is suitable for intestinal mitochondrial related disease groups and normal intestinal disease prevention groups.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the application is as follows:

[0007] In a first aspect, the application provides a preparation method of acetylated hawthorn polysaccharide, comprising the following steps:

[0008] The fresh hawthorn fruit is homogenized in a low eutectic solvent, and is extracted by a physical method to obtain an extract;

[0009] The extract is subjected to alcohol precipitation and centrifugation to obtain crude polysaccharide;

[0010] The crude polysaccharide is redissolved in water, and is subjected to deproteinization, concentration, dialysis and freeze-drying to obtain acetylated hawthorn polysaccharide;

[0011] The acetylation degree of the acetylated hawthorn polysaccharide is 0.25% to 0.40%.

[0012] The inventors found in the research process that, compared with hawthorn dry products, hawthorn powder and other raw materials, the fresh hawthorn fruit has complete cell structure, undamaged endogenous enzyme system and undegraded active ingredients. The acetylated polysaccharide in the fresh hawthorn fruit exists in a natural and complete state in the cell wall or intracellularly. The mild low eutectic solvent (DES) extraction (such as a choline-lactic acid system) can destroy the cell structure while avoiding strong heat and drastic pH changes, thereby maximizing the protection of acetyl groups.

[0013] In addition, the physical method and the low eutectic solvent realize synergistic effect through close cooperation of “wall breaking” and “dissolution”. The physical method uses mechanical forces such as cavitation effect to first destroy the hawthorn cell wall structure, creating microporous channels, not only reducing the mass transfer resistance, but also opening up channels for the penetration of the low eutectic solvent; and the low eutectic solvent relies on its designable hydrogen bond network to further disintegrate the cell wall and efficiently dissolve and release the acetylated polysaccharide, and its mild characteristics can also protect the heat-sensitive acetyl groups. The two form a dynamic enhancement cycle: the physical treatment strengthens the penetration and action of the solvent, and the solvent penetration makes the internal structure more sensitive to the physical field, thereby realizing efficient and high-quality extraction of the acetylated hawthorn polysaccharide in a short time.

[0014] In some other embodiments, 2 to 5 mL of the low eutectic solvent is added per gram of the fresh hawthorn fruit;

[0015] The homogenization power is 400~600 W, and the homogenization time is 5~15 min.

[0016] Specifically, add 2, 3, 4, or 5 mL of eutectic solvent per gram of fresh hawthorn fruit. Precisely controlling the ratio of fresh hawthorn fruit to eutectic solvent within this range ensures that DES has sufficient volume to fully surround, wet, and penetrate the hawthorn pulp cells, while maintaining a sufficient concentration gradient as a driving force to enable the polysaccharides to dissolve efficiently and rapidly.

[0017] The homogenization process uses a power of 400, 500, or 600 W and a homogenization time of 5, 10, or 15 minutes. Homogenizing fresh hawthorn fruit with a eutectic solvent to break down the cell walls is highly beneficial for subsequent physical impregnation (ultrasound, microwave) enhanced extraction, making the entire process smoother and more controllable.

[0018] In some other embodiments, the eutectic solvent comprises a hydrogen bond donor and a hydrogen bond acceptor, wherein the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is (1~5):(1~5).

[0019] The hydrogen bond donor is choline chloride, and the hydrogen bond acceptor is one or more of ethylene glycol, lactic acid, urea, and glucose.

[0020] Specifically, the molar ratio of hydrogen bond donor to hydrogen bond acceptor is 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 3:1, 4:1, 5:1, 3:2, 3:4, or 3:5. More specifically, the eutectic solvent is choline chloride-ethylene glycol, choline chloride-lactic acid, choline chloride-urea, or choline chloride-glucose; wherein the molar ratio of choline chloride-ethylene glycol, choline chloride-lactic acid, choline chloride-urea, or choline chloride-glucose is 2:1.

[0021] Studies have found that water or organic solvents have limited ability to dissolve cell wall components (cellulose, hemicellulose, pectin), resulting in low cell wall disruption efficiency. While strong acids, strong alkalis, or high temperatures are effective, they are highly destructive and lack selectivity. In contrast, eutectic solvents (DES) can preferentially soften and dissolve pectin and hemicellulose in the cell wall through strong hydrogen bonding, significantly weakening the structural integrity of the cell wall. Furthermore, based on the pre-softening of the cell wall by DES, the physical force of mechanical homogenization can more easily and thoroughly break down cells, greatly improving cell wall disruption efficiency and releasing intracellular polysaccharides.

[0022] In some other implementations, the physical method is one or more of multi-frequency ultrasound, microwave, and ultra-high voltage.

[0023] The frequency of the multi-frequency ultrasound is 25~75 kHz, and the duration of the multi-frequency ultrasound is 10~30 min.

[0024] The microwave power is 700~900 W, and the microwave duration is 10~30 min;

[0025] The ultra-high pressure is 500~700 MPa, and the ultra-high pressure time is 5~20 min.

[0026] Specifically, the frequency of the multi-frequency ultrasound is at least two of 25, 35, 45, 55, 65 or 75 kHz, and the duration of the multi-frequency ultrasound is 10, 15, 20, 25 or 30 min. The multi-frequency ultrasound treatment and the eutectic solvent achieve synergistic effect through the close cooperation of "cell wall breaking" and "dissolving", thereby improving the polysaccharide yield.

[0027] The microwave power is 700, 800, or 900 W, and the microwave duration is 10, 15, 20, 25, or 30 minutes. Microwave extraction is a highly efficient, rapid, energy-saving, environmentally friendly, and easily automated extraction technology. When microwaves are used in conjunction with novel green solvents such as DES, they can exhibit significant synergistic advantages, making them an ideal choice for preparing highly active plant extracts.

[0028] The ultra-high pressure is applied at pressures of 500, 600, or 700 MPa for 5, 10, 15, or 20 minutes. Ultra-high pressure utilizes extremely high hydrostatic pressure to physically compress and destroy cell structures at room temperature, forcing the cell contents to be released instantaneously into the solvent.

[0029] In some other embodiments, alcohol precipitation is performed using ethanol, the precipitation temperature is 2-5°C, and the precipitation time is 10-24 h.

[0030] The volume concentration of ethanol is 10% to 40%.

[0031] Specifically, the temperature for alcohol precipitation is 2, 3, 4 or 5°C, the precipitation time is 10, 12, 14, 16, 18, 20, 22 or 24 h, and the volume concentration of ethanol is 10%, 15%, 20%, 25%, 30%, 35% or 40%.

[0032] This invention does not introduce acetyl groups from the outside; rather, the acetyl groups are inherent in hawthorn itself. Specifically, during the synthesis of the cell wall (especially the pectin component) of hawthorn fruit, endogenous acetyltransferases catalyze the transfer of acetyl groups from acetyl-CoA to specific hydroxyl groups (such as the O-2 or O-3 positions) of the polysaccharide chain (especially galacturonic acid). The above treatment introduces hydrophobic acetyl groups into hawthorn polysaccharides, reducing the overall hydrophilicity of the polysaccharide molecules and disrupting the stable hydrogen bond network formed with water molecules. This significantly reduces the "solubility" of the polysaccharide in an ethanol-water mixture. At low alcohol concentrations, the effect of ethanol as a "poor solvent" is amplified, making it easier to damage the already fragile solvation layer, leading to the contraction, aggregation, and eventual precipitation of the polysaccharide molecular chains.

[0033] Secondly, the present invention provides acetylated hawthorn polysaccharide prepared by the method described in the first aspect.

[0034] Hawthorn polysaccharides are polymeric sugars composed of monosaccharides such as galacturonic acid, arabinose, galactose, and rhamnose. Among them, galacturonic acid is easily modified by groups such as acetyl groups. The mechanism may be that the acetylation potential provided by the extraction system itself (DES components) and the raw materials themselves (endogenous precursors and enzymes in fresh hawthorn fruit) provides the energy required for the reaction through physical fields (heat, ultrasound, etc.), thereby completing the extraction and mild acetylation modification simultaneously in an integrated green process, which is in line with the strategy of green chemistry principles.

[0035] In some other embodiments, the degree of acetylation of the acetylated hawthorn polysaccharide is 0.25% to 0.40%.

[0036] This invention modifies polysaccharides by "in situ acetylation" or "endogenous acetylation" without adding traditional acetylation reagents such as acetic anhydride or acetic acid, thereby obtaining acetyl groups with different contents and substitution sites, changing the molecular weight, monosaccharide composition and spatial structure of polysaccharides, and thus improving the biological activity of polysaccharides.

[0037] Thirdly, the acetylated hawthorn polysaccharide described in the second aspect of the present invention has one or more of the following applications:

[0038] (1) Application in the preparation of products for the prevention and / or treatment of intestinal mitochondrial dysfunction;

[0039] (2) Application in the preparation of products that enhance intestinal mitochondrial membrane potential;

[0040] (3) Application in the preparation of products that improve intestinal mitochondrial dynamics imbalance;

[0041] (4) Application in the preparation of products that improve intestinal mitochondrial autophagy;

[0042] (5) Application in the preparation of products that improve the activity of the intestinal respiratory chain complex;

[0043] (6) Application in the preparation of products that improve the production of adenosine triphosphate in the intestine.

[0044] In some other embodiments, intestinal mitochondrial dysfunction is a decrease in mitochondrial membrane potential, mitochondrial dynamic imbalance, or insufficient energy supply; the intestine is the colon, cecum, or small intestine.

[0045] Improving intestinal mitochondrial dynamics imbalance involves increasing the expression of mitochondrial fusion factors and decreasing the expression of mitochondrial fission factors. The mitochondrial fusion factors are specifically outer membrane fusion proteins (MFN1 / MFN2) and inner membrane fusion proteins (OPA1). The mitochondrial fission factors are specifically mitochondrial dynamics-associated protein 1 (DRP1), fission protein 1 (FIS1), and fission factor (MFF).

[0046] Improving intestinal mitophagy involves regulating the expression levels of key mitophagy proteins. These key mitophagy proteins are phosphatase and tensin homolog gene-induced kinase 1 (PINK1), Parkin, nucleosome assembly protein 1-like 1 (NAP1), and mitochondrial stress-induced protein (SINTBAD).

[0047] Improving the activity of the intestinal respiratory chain complex involves regulating the activity level of the respiratory chain complex; the respiratory chain complex specifically consists of complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome c reductase), complex IV (cytochrome c oxidase), and complex V (ATP synthase).

[0048] Improving intestinal adenosine triphosphate (ATP) production aims to increase the rate and level of ATP synthesis. Key components involved in ATP production include adenylate translocase (ANT), ATP synthase (complex V), creatine phosphokinase (CK), and adenylate kinase (AK).

[0049] In some other embodiments, the product is one of a drug, health product, food, or preparation.

[0050] Acetylated hawthorn polysaccharide can be used alone or in combination with other ingredients in functional foods and health supplements. In clinical applications, it is used as a food component in medical foods or health supplements to improve intestinal mitochondrial function. It can also be formulated using conventional pharmaceutical processes, either alone or in combination with other drugs, into different dosage forms suitable for clinical use. For example, excipients, flavoring agents, and preservatives can be added to create anti-enteritis capsules or probiotic oral liquids. Dosage forms include capsules, oral liquids, injections, tablets, powders, and granules.

[0051] The beneficial effects of this invention are:

[0052] (1) This invention cleverly utilizes the solvent properties of eutectic solvents, the enhanced transmission and reaction capabilities of physical fields, and the precipitation properties of acetylated polysaccharides. Through the synergistic effect of these three factors, a method for "extraction-modification integration" of acetylated hawthorn polysaccharides is constructed. This method has high extraction efficiency, simple operation, low cost, and is suitable for industrial production.

[0053] (2) In the preparation method of the present invention, the physical method destroys the cell wall structure of hawthorn by mechanical force, which greatly promotes the penetration and mass transfer of the eutectic solvent; the strong hydrogen bond network of the eutectic solvent can not only efficiently extract acetylated hawthorn polysaccharide, but also catalyze the acetylation reaction itself or as a medium, so as to realize the one-pot method of "extraction-modification"; and the subsequent low concentration ethanol precipitation utilizes the characteristics of the acetylated polysaccharide to enhance hydrophobicity and precipitate at low alcohol concentration, so as to realize the efficient and mild separation and preliminary purification of the modified polysaccharide.

[0054] (3) The acetylated hawthorn polysaccharide prepared by this invention has obvious acetylation group signal, and has the activity of regulating intestinal mitochondrial homeostasis in vitro and in vivo, and has no toxic side effects. It is suitable not only for people with intestinal mitochondrial related diseases, but also for the prevention of intestinal diseases in normal people. Attached Figure Description

[0055] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0056] Figure 1 This is a scanning electron microscope image of acetylated hawthorn polysaccharide in Example 1 of the present invention;

[0057] Figure 2 This is a chromatogram of the molecular weight of acetylated hawthorn polysaccharide in Example 1 of the present invention;

[0058] Figure 3 The Fourier transform infrared spectrum of acetylated hawthorn polysaccharide in Example 1 of this invention;

[0059] Figure 4 This is the 1H NMR spectrum of acetylated hawthorn polysaccharide in Example 1 of the present invention;

[0060] Figure 5The diagram shows the effect of acetylated hawthorn polysaccharide on mitochondrial function in Example 1 of this invention. A represents the effect of acetylated hawthorn polysaccharide on the mitochondrial membrane potential of Caco-2 intestinal epithelial cells induced by lipopolysaccharide; B represents the effect of acetylated hawthorn polysaccharide on the relative expression level of mitochondrial dynamics-related protein 1 in the colon of mice induced by a high-fat diet; C represents the effect of acetylated hawthorn polysaccharide on the relative expression level of kinase 1 induced by phosphatase and tensin homolog genes in the colon of mice induced by a high-fat diet; D represents the effect of acetylated hawthorn polysaccharide on the activity of mitochondrial respiratory chain complex I in the colon of mice induced by a high-fat diet; and E represents the effect of acetylated hawthorn polysaccharide on the production of adenosine triphosphate (ATP) in the colon of mice induced by a high-fat diet. Detailed Implementation

[0061] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Specific conditions not specified in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Components whose manufacturers are not specified are all commercially available conventional products.

[0062] The chemical preparation of acetylated polysaccharides suffers from problems such as easy structural damage, low product purity and uniformity, and significant environmental impact. This invention addresses these issues by using specific fresh hawthorn fruit as raw material. Through physical extraction combined with a eutectic solvent and low-concentration ethanol precipitation, without the need for acetylation reagents like heparin, it achieves "extraction + modification" of hawthorn polysaccharides, yielding highly acetylated hawthorn polysaccharides. Furthermore, the mechanism by which acetylated polysaccharides regulate intestinal mitochondrial homeostasis is investigated.

[0063] The present invention will be further described below with reference to the embodiments.

[0064] Example 1

[0065] This embodiment provides a method for preparing acetylated hawthorn polysaccharide by multi-frequency ultrasound combined with eutectic solvent extraction, specifically including the following steps:

[0066] (1) Take 10 kg of fresh hawthorn fruit and add it to a reaction vessel. Add 30 L of eutectic solvent (choline chloride is the hydrogen bond acceptor, lactic acid is the hydrogen bond donor, and the molar ratio of choline chloride to lactic acid is 2:1). Homogenize the mixture for 10 min using a 500 W blender. Extract the mixture by sonication at multiple frequencies (25 kHz, 45 kHz, and 75 kHz in sequence) for 20 min. After extraction, centrifuge at 10000 r / min for 10 min, retain the supernatant, and cool to room temperature.

[0067] (2) Add 20% volume concentration of anhydrous ethanol to the supernatant, stir at 500 r / min for 15 min, precipitate overnight at 4℃, and centrifuge at 10000 r / min for 10 min to obtain the precipitate, which is crude polysaccharide.

[0068] (3) After redissolving in distilled water, the protein was removed by the Sevage method, and the mixture was concentrated by rotary evaporation until there was no organic reagent odor. After appropriate concentration, the mixture was dialyzed for 72 h and freeze-dried to produce sugar powder (yield of 6.75%), thus obtaining acetylated hawthorn polysaccharide with a purity of 97.6%.

[0069]

[0070] The acetylated hawthorn polysaccharide molecule was simplified to a D-glucanopyranose chain, and the degree of acetylation of the hawthorn polysaccharide was determined by hydrolysis. The results showed that the degree of acetylation of the acetylated hawthorn polysaccharide was 0.39%.

[0071] The particle morphology of acetylated hawthorn polysaccharide was characterized using scanning electron microscopy. The specific implementation scheme is as follows:

[0072] The particle morphology of acetylated hawthorn polysaccharide was observed using a scanning electron microscope (JSM-6380LV, JEOL, Japan). The samples were dried in a hot air drying oven at 105℃ for 5 h, fixed to the sample preparation stage with double-sided tape under infrared light, and then sputtered with gold. Finally, they were detected at a 10 kV high-speed voltage. The results are as follows: Figure 1 As shown. By Figure 1 It is evident that the acetylated hawthorn polysaccharide sample exhibits a loose, porous, amorphous, layered structure. It is composed of stacked and interconnected thin sheets of varying sizes and irregular shapes, forming a complex three-dimensional network. This unique layered, porous structure is likely due to the combined effects of hydrogen bonding between polysaccharide molecular chains and the interactions between newly introduced acetyl groups.

[0073] The monosaccharide composition analysis of acetylated hawthorn polysaccharides was performed using chromatographic techniques. The core procedure involved first completely acid-hydrolyzing the acetylated hawthorn polysaccharides into individual monosaccharide molecules, and then separating, identifying, and quantifying the hydrolysis products. The specific implementation plan is as follows:

[0074] The glycosidic bonds of acetylated hawthorn polysaccharides were completely broken at high temperature using trifluoroacetic acid or sulfuric acid to obtain a mixture of monosaccharides. These monosaccharide molecules were then derivatized using 1-phenyl-3-methyl-5-pyrazolone (PMP). Finally, the derivatized monosaccharides were separated using techniques such as high-performance liquid chromatography (HPLC) or gas chromatography-mass spectrometry (GC-MS). By comparing the retention time and characteristic ion fragments with those of standard monosaccharides, the various monosaccharide types constituting acetylated hawthorn polysaccharides were accurately identified, and their molar proportions were calculated based on peak areas, ultimately determining the monosaccharide composition of acetylated hawthorn polysaccharides. The results are shown in Table 1.

[0075] Table 1 Monosaccharide composition of acetylated hawthorn polysaccharide

[0076]

[0077] Table 1 shows that the monosaccharide composition of acetylated hawthorn polysaccharides is glucose (46.9%), galacturonic acid (35.8%), arabinose (11.0%), rhamnose (2.2%), galactose (1.9%), fucose (0.9%), xylose (0.7%), and glucuronic acid (0.6%). Among these, galacturonic acid is easily modified by acetyl groups. Hawthorn polysaccharides themselves contain acetyl groups, a result of their natural biosynthesis within the plant. Specifically, during the synthesis of the cell wall (especially the pectin component) of hawthorn fruit, endogenous acetyltransferases catalyze the transfer of acetyl groups from acetyl-CoA to specific hydroxyl groups (such as the O-2 or O-3 positions) on the polysaccharide chain (especially galacturonic acid). This natural acetylation modification is a common plant physiological phenomenon, whose main function is to regulate the structure and properties of the cell wall: by introducing acetyl groups to generate steric hindrance, it prevents the formation of overly dense and rigid hydrogen bond networks or calcium ion cross-linking between polysaccharide molecules, thereby helping to maintain the flexibility and porosity of fruit tissue.

[0078] The molecular weight of acetylated hawthorn polysaccharides was determined using high-performance size exclusion chromatography-multi-angle light scattering (PSLC). The specific implementation scheme is as follows:

[0079] Acetylated hawthorn polysaccharide samples dissolved in dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) were injected into a chromatographic column filled with porous gel. Based on the different hydrodynamic volumes, molecules of different sizes were separated in the column. The eluted components were first analyzed for concentration using a differential refractive index detector (RI), and their absolute molecular weight was directly detected by a multi-angle light scattering (MALS) spectrometer. Figure 2The chromatogram of the molecular weight of acetylated hawthorn polysaccharide was generated using raw data collected by Astra8 software based on high performance size exclusion chromatography-multi-angle laser light scattering-differential detector (HPSEC-MALS-RID). The red curve in the figure represents the chromatographic response signal of the sample, with the horizontal axis representing elution time (min) and the vertical axis representing relative scale (%), reflecting the relative intensity of each eluted component.

[0080] from Figure 2 As can be seen, the sample exhibits a major elution peak within the range of approximately 12-20 min, with higher signal intensity in the 14-18 min interval, indicating concentrated elution and a relatively concentrated molecular weight distribution during this period. The overall spectrum displays a typical single-peak distribution, suggesting a relatively uniform molecular weight distribution. Utilizing the direct relationship between light scattering intensity and molecular weight, combined with the sample concentration at each time point, the absolute molecular weight of each eluted component (at each time point in the figure) can be calculated. Finally, by weighted averaging the molecular weights at each point within the entire elution peak range, the weight-average molecular weight (Mw) of the sample, 214 kDa, can be obtained. The average molecular weight (Mn) is 140 kDa, and the maximum molecular weight (Mp) is 173 kDa.

[0081] The infrared spectrum of acetylated hawthorn polysaccharide was determined using a Fourier transform infrared spectroscopy scanner. The specific implementation plan is as follows:

[0082] The dried acetylated hawthorn polysaccharide sample was mixed with potassium bromide at a ratio of 1:100 to 1:150 and thoroughly ground. The ground powder was poured into a mold and vacuum-pressed into tablets at 12 MPa for 30 seconds until transparent. The scanning range of the Fourier transform infrared spectroscopy scanner was 4000–400 cm⁻¹. -1 The resolution is 4 cm. -1 The number of scans ranged from 16 to 32. The test results are as follows: Figure 3 As shown. By Figure 3 It can be known that 2935.18 cm -1 The strong absorption peak at 1715.68 cm⁻¹ is due to the stretching vibration of the methyl group in the acetyl group. -1 The strong absorption peak at this point is due to the stretching vibration of the carbonyl group in the acetyl group. This indicates that the acetylated hawthorn polysaccharide structure contains an acetyl group.

[0083] The degree of acetyl groups in acetylated hawthorn polysaccharides was analyzed using nuclear magnetic resonance spectroscopy. The specific implementation scheme is as follows:

[0084] The acetylated hawthorn polysaccharide sample was fully dissolved in deuterated dimethyl sulfoxide or deuterated water. Proton NMR spectroscopy (¹H NMR) was performed using a Bruker AVANCE NEO 800 MHz superconducting NMR spectrometer, with an operating frequency of [insert frequency here].1 H 800MHz. (By proton spectrum) 1 The intensity of the acetylation characteristic peak in 1H NMR was used to qualitatively analyze the acetylation results of acetylated hawthorn polysaccharides. The results are as follows: Figure 4 As shown. By Figure 4 It is known that acetylated hawthorn polysaccharide has a distinct acetyl hydrogen characteristic peak at 2.2 ppm.

[0085] Caco-2 intestinal epithelial cells and C57BL / 6J mice were selected. Endotoxin and a high-fat diet were used to induce mitochondrial dysfunction in cells and animals, respectively. The effects of acetylated hawthorn polysaccharide on intestinal mitochondrial function were investigated. The specific implementation plan is as follows:

[0086] The Caco-2 intestinal epithelial cell growth medium was DMEM medium, with 10% fetal bovine serum (FBS), 100 U / mL penicillin and 100 U / mL streptomycin added. The culture conditions were set at 5% CO2 and 37℃.

[0087] When the Caco-2 intestinal epithelial cells reach approximately 90% confluence, the cells are fed at a rate of 1×10⁻⁶. 5 Cells were seeded in 96-well plates and divided into three groups: a control group (DMEM medium), a model group (DMEM medium), and an acetylated hawthorn polysaccharide group (DMEM medium with acetylated hawthorn polysaccharide concentration of 50 μg / mL). After 24 h of culture, the medium was replaced with fresh serum-free medium and cultured for another 4 h. Then, LPS (200 ng / mL) was added to both the model and intervention groups, and the cells were incubated in a CO2 incubator for another 24 h. Subsequently, the cells were digested with trypsin cell digestion solution for 2.5 min to prepare a cell suspension, and 0.5 mL of the suspension was transferred to centrifuge tubes. The suspensions were then centrifuged at 400×10⁻⁶ at room temperature. g Centrifuge for 5 min to remove supernatant, then resuspend cells in JC-1 working solution. Incubate at 4°C for 15 min in a cell culture incubator. Incubate at 400× at room temperature. g Centrifuge for 5 min and remove the supernatant. Resuspend the cells in 2 mL of PBS buffer, centrifuge to remove the supernatant, and repeat once. Resuspend the cells in 0.5 mL of PBS and analyze the mitochondrial membrane potential using immunofluorescence staining.

[0088] A mouse model of intestinal mitochondrial dysfunction was established using a high-fat diet. Eight-week-old male C57BL / 6J mice were selected and, after one week of acclimatization, randomly divided into five groups of ten mice each: a control group (fed standard diet), a model group (fed a 60% high-fat diet), and an acetylated hawthorn polysaccharide treatment group (fed a high-fat diet plus gavage administration of 900 mg / kg·bw acetylated hawthorn polysaccharide). The experiment lasted for 12 weeks, during which animals had free access to food and water. Western blotting was used to determine the protein expression levels of mitochondrial dynamism-associated protein 1 (DRP1) and phosphatase and tensin homolog-induced kinase 1 (PINK1) in the mouse colon. The activity of the mitochondrial respiratory chain complex I (specifically complex I) and ATP production in the mouse colon were measured using a mitochondrial respiratory chain complex I activity assay kit and an adenosine triphosphate (ATP) fluorescence assay kit, respectively. Results are shown below. Figure 5 As shown, A represents the effect of acetylated hawthorn polysaccharide on lipopolysaccharide-induced mitochondrial membrane potential in Caco-2 intestinal epithelial cells; B represents the effect of acetylated hawthorn polysaccharide on the relative expression level of mitochondrial dynamism-associated protein 1 (DRP1) in the colon of mice induced by a high-fat diet; C represents the effect of acetylated hawthorn polysaccharide on the relative expression level of phosphatase and tensin homolog-induced kinase 1 (PINK1) in the colon of mice induced by a high-fat diet; D represents the effect of acetylated hawthorn polysaccharide on the activity of mitochondrial respiratory chain complex I in the colon of mice induced by a high-fat diet; and E represents the effect of acetylated hawthorn polysaccharide on the production of adenosine triphosphate (ATP) in the colon of mice induced by a high-fat diet. p <0.05 is considered significant. p <0.01 indicates highly significant; compared with the model group, # p <0.05 is considered significant. p <0.01 indicates highly significant.

[0089] like Figure 5 As shown in Figure A, compared with the control group, the red fluorescence of Caco-2 cells in the model group was weakened, while the green fluorescence was enhanced. Compared with the model group Caco-2 cells, the red fluorescence of Caco-2 cells was enhanced and the green fluorescence was weakened after intervention with acetylated hawthorn polysaccharide, indicating an increase in mitochondrial membrane potential. DRP1 is an important mitochondrial fission protein. Figure 5 Results B showed that the DRP1 protein content in the colon of mice after acetylated hawthorn polysaccharide intervention was significantly lower than that in the model group, indicating that acetylated hawthorn polysaccharide improved mitochondrial dynamics imbalance. PINK1 is a classic pathway protein mediating mitophagy. Figure 5The results showed that the PINK1 protein content in the colon of mice treated with acetylated hawthorn polysaccharide was significantly lower than that in the model group, indicating that acetylated hawthorn polysaccharide improved mitophagy. Respiratory chain complex I is a key "engine" and "start switch" on the inner mitochondrial membrane; its activity directly determines mitochondrial energy conversion efficiency, reactive oxygen species levels, and overall health. Figure 5 As shown in D, after intervention with acetylated hawthorn polysaccharide, the activity of mitochondrial respiratory chain complex 1 in the mouse colon was significantly increased compared with that in the model group, indicating that acetylated hawthorn polysaccharide improved its mitochondrial respiratory chain complex activity. Mitochondria are the main site of ATP production, and ATP production is a core indicator for measuring mitochondrial function. Figure 5 As shown in Figure E, acetylated hawthorn polysaccharide intervention significantly increased ATP production in the mouse colon, indicating that acetylated hawthorn polysaccharide enhanced ATP production. In summary, these results demonstrate that acetylated hawthorn polysaccharide improved intestinal mitochondrial dysfunction.

[0090] Example 2

[0091] This embodiment provides a microwave-assisted eutectic solvent extraction method for preparing acetylated hawthorn polysaccharide, specifically including the following steps:

[0092] (1) Take 10 kg of fresh hawthorn fruit and add it to the reaction vessel. Add 30 L of eutectic solvent (the hydrogen bond acceptor is choline chloride, the hydrogen bond donor is ethylene glycol, and the molar ratio of choline chloride to ethylene glycol is 3:1). Homogenize the mixture for 10 min using a 500 W blender. Microwave (800 W) for 30 min. After extraction, centrifuge at 10000 r / min for 10 min, retain the supernatant, and cool to room temperature.

[0093] (2) Add 20% volume of anhydrous ethanol and stir rapidly at 500 r / min for 15 min. Precipitate overnight at 4℃ and centrifuge at 10000 r / min for 10 min to obtain the precipitate, which is the crude polysaccharide.

[0094] (3) After redissolving in distilled water, the protein was removed by the Sevage method, and the mixture was concentrated by rotary evaporation until there was no organic reagent odor. After appropriate concentration, the mixture was dialyzed for 72 h and freeze-dried to produce sugar powder (yield of 6.17%), which yielded acetylated hawthorn polysaccharide with a purity of 96.3% and a degree of acetylation of 0.31%.

[0095] Example 3

[0096] This embodiment provides a method for preparing acetylated hawthorn polysaccharide by ultra-high pressure synergistic eutectic solvent extraction, specifically including the following steps:

[0097] (1) Take 10 kg of fresh hawthorn fruit and add it to the reaction vessel. Add 30 L of eutectic solvent (the hydrogen bond acceptor is choline chloride, the hydrogen bond donor is glycerol, and the molar ratio of choline chloride to glycerol is 1:2). Homogenize the mixture for 10 min using a 500 W blender. Treat with ultra-high pressure (600 MPa) for 20 min. After extraction, centrifuge at 10000 r / min for 10 min, retain the supernatant, and cool to room temperature.

[0098] (2) Add 20% volume of anhydrous ethanol and stir rapidly at 500 r / min for 15 min. Precipitate overnight at 4℃ and centrifuge at 10000 r / min for 10 min to obtain the precipitate, which is the crude polysaccharide.

[0099] (3) After redissolving in distilled water, the protein was removed by the Sevage method, and the mixture was concentrated by rotary evaporation until there was no organic reagent odor. After appropriate concentration, the mixture was dialyzed for 72 h and freeze-dried to produce sugar powder (yield of 6.34%), which yielded acetylated hawthorn polysaccharide with a purity of 96.8% and a degree of acetylation of 0.34%.

[0100] Comparative Example 1

[0101] This comparative example provides a water extraction and alcohol precipitation method for preparing acetylated hawthorn polysaccharide, specifically including the following steps:

[0102] (1) Take 10 kg of fresh hawthorn fruit and add it to the reaction vessel. Add 30 L of distilled water and homogenize it for 10 min using a 500 W blender. Set the temperature of the reaction vessel to 90℃. After reaching the temperature, keep it warm for 120 min. After the holding time is up, release the material. After the extraction is complete, centrifuge at 10000 r / min for 10 min, retain the supernatant, and cool it to room temperature.

[0103] (2) Add 75% volume of anhydrous ethanol and stir rapidly at 500 r / min for 15 min. Incubate overnight at 4℃ for alcohol precipitation, and centrifuge at 10000 r / min for 10 min to obtain the precipitate, which is the crude polysaccharide.

[0104] (3) After redissolving in distilled water, the protein was removed by the Sevage method, and the mixture was concentrated by rotary evaporation until there was no organic reagent odor. After appropriate concentration, the mixture was dialyzed for 72 h and freeze-dried to produce sugar powder (yield of 1.34%), which yielded acetylated hawthorn polysaccharide with a purity of 91.6% and a degree of acetylation of 0.07%.

[0105] Compared with Example 1, Comparative Example 1 used distillation as the extraction agent, did not undergo multi-frequency ultrasonic treatment, and used high-concentration ethanol for extraction (75% by volume anhydrous ethanol), resulting in a lower yield and degree of acetylation of the produced sugar powder. This indicates that multi-frequency ultrasonic treatment and eutectic solvent achieve synergistic effects through the close combination of "cell wall disruption" and "dissolution," thereby improving the yield of sugar powder. For hydrophilic unmodified acetyl polysaccharides, a large amount of ethanol (high alcohol concentration) is required to significantly weaken hydration, destroy their stable hydrated shell, and make the interaction between polysaccharide molecular chains stronger than the interaction between the chains and the solvent, thus causing precipitation.

[0106] Comparative Example 2

[0107] Unlike Example 1, in step (1), extraction was performed using ultrasonic treatment at a frequency of 45 kHz for 20 min. Other preparation methods were the same as in Example 1.

[0108] The sugar was made into powder (yield of 5.17%), which yielded acetylated hawthorn polysaccharide with a purity of 95.6% and a degree of acetylation of 0.27%.

[0109] Compared with Example 1, Comparative Example 2, which used a single frequency of ultrasound treatment, resulted in a lower yield and degree of acetylation of the produced sugar powder. This indicates that multi-frequency ultrasound, through the synergistic effect between frequencies, creates a more uniform, denser, and more intense cavitation field, thus comprehensively surpassing single-frequency ultrasound in mechanism. It can more efficiently disrupt cell structure and enhance mass transfer processes, thereby achieving a higher polysaccharide extraction rate in a shorter time and with lower energy consumption. Furthermore, it may reduce polymer degradation caused by prolonged ultrasound treatment, better preserving the natural structure and bioactivity of polysaccharides.

[0110] Comparative Example 3

[0111] Unlike Example 1, in step (1), acetic anhydride was used to replace the eutectic solvent and the physical method, while the other preparation methods were the same as in Example 1.

[0112] The sugar was made into powder (yield of 3.29%), which yielded acetylated hawthorn polysaccharide with a purity of 95.2% and a degree of acetylation of 0.21%.

[0113] Compared with Example 1, Comparative Example 3, which prepared acetylated hawthorn polysaccharide by adding acetic anhydride using a chemical method, showed lower yield and degree of acetylation. This indicates that although the chemical method can achieve the basic acetylation reaction, its reaction process relies on random molecular collisions, resulting in localized uneven concentrations and low mass transfer efficiency. This leads to uneven distribution of acetyl groups and limited overall substitution. Furthermore, the relatively vigorous homogeneous reaction conditions may damage some of the higher-order structures of the polysaccharide, causing molecular chain breakage and significantly reducing the yield of the final product. In contrast, the multi-frequency ultrasound-assisted technology used in Example 1, through the microjets generated by cavitation and strong stirring, achieved extreme mixing and directional enhanced mass transfer in the reaction system. This not only more efficiently activated the polysaccharide molecules and protected the acetyl groups but also effectively shortened the reaction time and reduced damage to the polysaccharide backbone. Thus, while maintaining high purity and degree of acetylation, it more comprehensively preserved the natural conformation and biological activity of the polysaccharide.

[0114] Comparative Example 4

[0115] Unlike Example 1, in step (1), hawthorn powder was used to replace fresh hawthorn fruit in equal amounts, while the other preparation methods were the same as in Example 1.

[0116] The sugar was made into powder (yield 6.01%), which yielded acetylated hawthorn polysaccharide with a purity of 94.5% and a degree of acetylation of 0.14%.

[0117] Compared with Example 1, Comparative Example 4, which prepared acetylated hawthorn polysaccharides from hawthorn powder, showed lower yield and degree of acetylation. This indicates that although the main chemical components of the raw materials are consistent, changes in physical morphology have a decisive impact on the final result. The drying and pulverizing process may lead to the collapse of hawthorn cell walls, loss of some active ingredients, and irreversible hydrogen bonding of polysaccharides, forming a denser structure that hinders the penetration of eutectic solvents (DES). In contrast, Example 1, which directly uses fresh hawthorn fruit, has a more complete cell structure and a full moisture content, which is more conducive to the penetration of DES and the transmission of ultrasonic cavitation effects. This allows for more efficient simultaneous dissolution, activation, and modification of components, resulting in significant advantages in both yield and degree of acetylation. This highlights the crucial role of the initial physical state of the raw materials in the success of the green preparation process.

[0118] Comparative Example 5

[0119] Unlike Example 1, in step (1), an equal amount of ethanol was used to replace the eutectic solvent, while the other preparation methods were the same as in Example 1.

[0120] The sugar was made into powder (yield of 3.15%), which yielded acetylated hawthorn polysaccharide with a purity of 95.1% and a degree of acetylation of 0.12%.

[0121] Compared with Example 1, Comparative Example 5, which used ethanol as an extractant to prepare acetylated hawthorn polysaccharide, showed lower yield and degree of acetylation. This indicates that although ethanol is a common extraction solvent for natural polysaccharides, its solvation ability mainly relies on breaking hydrogen bonds, and it cannot effectively disrupt the dense structure of plant cell walls to achieve sufficient dissolution like a eutectic solvent (DES). The high molecular diffusion resistance in the ethanol system severely limits the mass transfer and grafting efficiency of acetyl groups onto the polysaccharide chains. In contrast, the eutectic solvent system used in Example 1 combines efficient cell wall disruption, excellent polysaccharide solubility, and inherent catalytic activity. Combined with the physical field, it creates a more favorable reaction environment for component dissolution and molecular modification, thus exhibiting significant advantages in yield, purity, and degree of acetylation. This demonstrates that the rational selection of the solvent system has a decisive impact on achieving efficient and green preparation.

[0122] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing acetylated hawthorn polysaccharide, characterized in that, Includes the following steps: Fresh hawthorn fruit was homogenized in a eutectic solvent and extracted using a physical method to obtain the extract. Each gram of fresh hawthorn fruit is added to 2-5 mL of a eutectic solvent; The homogenization power is 400~600 W, and the homogenization time is 5~15 min; The eutectic solvent includes a hydrogen bond donor and a hydrogen bond acceptor, wherein the molar ratio of the hydrogen bond donor and the hydrogen bond acceptor is (1~5):(1~5). The hydrogen bond donor is choline chloride, and the hydrogen bond acceptor is one or more of ethylene glycol, lactic acid, urea, and glucose. The physical method is one or more of the following: multi-frequency ultrasound, microwave, and ultra-high pressure. The frequency of the multi-frequency ultrasound is 25~75 kHz, and the duration of the multi-frequency ultrasound is 10~30 min. The power of the microwave is 700~900 W, and the duration of the microwave is 10~30 min; The ultra-high pressure is 500~700 MPa, and the ultra-high pressure time is 5~20 min; The extract was subjected to alcohol precipitation and centrifugation to obtain crude polysaccharide; The alcohol precipitation is performed using ethanol, with a precipitation temperature of 2-5℃ and a precipitation time of 10-24 h. The volume concentration of the ethanol is 10% to 40%; The crude polysaccharide was reconstituted with water, and then subjected to deproteinization, concentration, dialysis and freeze-drying to obtain acetylated hawthorn polysaccharide; The degree of acetylation of the acetylated hawthorn polysaccharide is 0.25% to 0.40%.

2. The acetylated hawthorn polysaccharide prepared by the method of claim 1.

3. The acetylated hawthorn polysaccharide according to claim 2, characterized in that, The degree of acetylation of the acetylated hawthorn polysaccharide is 0.25% to 0.40%.

4. An acetylated hawthorn polysaccharide according to claim 2 or 3, having the following applications: (1) Application in the preparation of products for the prevention and / or treatment of intestinal mitochondrial dysfunction.

5. An acetylated hawthorn polysaccharide according to claim 2 or 3, having one or more of the following applications: (1) Application in the preparation of products that enhance intestinal mitochondrial membrane potential; (2) Application in the preparation of products that improve intestinal mitochondrial dynamics imbalance; (3) Application in the preparation of products that improve intestinal mitochondrial autophagy; (4) Application in the preparation of products that improve the activity of the intestinal respiratory chain complex; (5) Application in the preparation of products that improve the production of adenosine triphosphate in the intestine.

6. The acetylated hawthorn polysaccharide according to claim 4, characterized in that, The intestinal mitochondrial dysfunction is characterized by decreased mitochondrial membrane potential, mitochondrial dynamic imbalance, or insufficient energy supply.

7. The acetylated hawthorn polysaccharide according to claim 5, characterized in that, The improvement of intestinal mitochondrial dynamics imbalance is achieved by increasing the expression of mitochondrial fusion factors and decreasing the expression of mitochondrial fission factors. The improvement of intestinal mitophagy involves regulating the expression levels of key mitophagy proteins; The improvement of intestinal respiratory chain complex activity is achieved by regulating the activity level of the respiratory chain complex. The improvement of intestinal adenosine triphosphate (ATP) production refers to increasing the synthesis rate and yield of ATP.

8. The acetylated hawthorn polysaccharide according to claim 4, characterized in that, The product is a type of medicine or health supplement.

9. The acetylated hawthorn polysaccharide according to claim 5, characterized in that, The product is a type of medicine or health supplement.

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