Preparation method and application of sugar acid-rich acanthopanax senticosus polysaccharide

Abstract

The application discloses a preparation method and application of a sugar acid-rich acanthopanax senticosus polysaccharide, and more particularly relates to the technical field of traditional Chinese medicines. The acanthopanax senticosus root is used as a raw material, a mechanical chemical activation combined with a microwave-assisted aqueous two-phase extraction technology is adopted, a pH fractionation precipitation method is combined to obtain acanthopanax senticosus crude polysaccharide, a GM-20 membrane is used to separate the polysaccharide to obtain a ten-thousand-grade polysaccharide ASAP, and DEAE-650M is used to purify the polysaccharide to obtain acanthopanax senticosus polysaccharide (ASAP-I). The preparation process has the characteristics of simple operation, high sugar acid content, uniform molecular weight, time saving, high efficiency, and green environmental protection. The obtained polysaccharide can reduce the active oxygen level in CL4176 nematodes, scavenge superoxide anions, inhibit the abnormal aggregation of A beta, delay the paralysis of nematodes induced by A beta, and enhance the stress resistance of nematodes, and can be applied to the preparation of medicines for nerve protection and stress resistance.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, and more specifically to a method for preparing and applying Acanthopanax senticosus polysaccharide rich in uronic acid. Background Technology

[0002] Acanthopanax senticosus ( Acanthopanax senticosus *Acanthopanax senticosus*, a plant belonging to the Araliaceae family, is used as a traditional tonic. Its dried rhizome is believed to invigorate qi and strengthen the spleen, tonify the kidneys, and calm the mind. It is commonly used in folk medicine for symptoms such as spleen and kidney yang deficiency, physical weakness, and loss of appetite. Chemical analysis shows that *Acanthopanax senticosus* contains various components including polysaccharides, glycosides, flavonoids, lignans, and coumarins, with polysaccharides being one of its main active ingredients. Modern pharmacological studies indicate that *Acanthopanax senticosus* polysaccharides possess antioxidant, anti-fatigue, immunomodulatory, and neuroprotective effects. Acidic polysaccharides rich in uronic acids such as glucuronic acid or galacturonic acid are important components of *Acanthopanax senticosus* polysaccharides. The free carboxyl groups in the uronic acid residues are core active groups for chelating metal ions and scavenging free radicals, playing a crucial role in anti-oxidative stress. Further rational development and utilization of *Acanthopanax senticosus* resources are needed, but problems such as low extraction rates of acidic polysaccharides and low uronic acid content remain unresolved.

[0003] In recent years, green extraction technologies have received widespread attention. Mechanochemical activation (mechanical ball milling) technology, by using mechanical force to disrupt the crystalline regions of plant cell walls, increases the specific surface area and reactivity of the raw materials, and has been successfully used for the pretreatment of various natural products. Aqueous two-phase extraction technology utilizes two water-soluble polymers or a polymer and salt to form a two-phase system in aqueous solution, enabling simultaneous extraction and preliminary purification of target components. Microwave-assisted extraction technology utilizes the rapid heating effect of microwaves to promote cell wall rupture and improve extraction efficiency. However, the technology of combining mechanochemical activation and microwave-assisted aqueous two-phase extraction for polysaccharide extraction has not yet been reported, especially in the field of Acanthopanax senticosus acidic polysaccharides. Furthermore, there are no reports of combining mechanochemical activation-microwave-assisted aqueous two-phase extraction with pH fractionation precipitation, membrane separation, or other techniques to achieve targeted enrichment of Acanthopanax senticosus acidic polysaccharides.

[0004] In recent years, with the continuous changes in the human living environment and the accelerated pace of life, people generally experience higher levels of stress, leading to negative emotions. Prolonged negative emotions can easily lead to serious psychological problems. The persistence of stressful situations can easily result in low mood, slowed thinking, and decreased memory for work and study. In severe cases, it can lead to decreased motor skills and stress tolerance, seriously impacting daily life and work. Different degrees of decreased motor and stress tolerance can lead to a series of health problems, such as falls due to poor coordination, slowed response to stimuli, and memory loss. As a traditional tonic, Acanthopanax senticosus has the effects of invigorating qi and strengthening the spleen, tonifying the kidneys and calming the mind. Modern pharmacological research shows that its polysaccharide components have significant antioxidant, anti-fatigue, and neuroprotective effects, effectively scavenging free radicals, alleviating oxidative stress, and enhancing the body's tolerance to adverse environments. Existing studies have demonstrated that Acanthopanax senticosus shows significant advantages in improving motor skills, enhancing stress response, and delaying functional decline, providing direction for future research or development of new drugs that regulate motor skills and stress tolerance.

[0005] Therefore, developing a method for preparing Acanthopanax senticosus polysaccharide with high extraction rate and uronic acid content, while also reducing ROS, scavenging superoxide anions and inhibiting Aβ aggregation activity, is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing Acanthopanax senticosus polysaccharide rich in uronic acid and its application in the preparation of neuroprotective agents.

[0007] This invention uses Acanthopanax senticosus root as raw material and employs a combination of mechanochemical activation and microwave-assisted aqueous two-phase extraction technology, along with pH-sequential precipitation, GM-20 membrane separation, and DEAE-650M purification, to obtain Acanthopanax senticosus polysaccharides. Mechanochemical activation (ball milling) disrupts the crystalline regions of the Acanthopanax senticosus cell wall, increasing the specific surface area. Then, selective extraction and simultaneous purification via a microwave-assisted aqueous two-phase system efficiently releases acidic polysaccharides while removing proteins and pigments in situ. Combined with pH gradient precipitation (pH 3.0, 5.0, 7.0), a specific fractionation strategy is designed to collect the pH 5.0 and 7.0 precipitates and discard the pH 3.0 precipitate, targeting the distribution pattern of acidic polysaccharides. This strategy fully leverages the synergistic advantages of mechanochemical activation pretreatment and aqueous two-phase extraction for targeted enrichment of acidic polysaccharides. The Acanthopanax senticosus polysaccharides of this invention can reduce ROS levels in CL4176 nematodes, scavenge superoxide anions, and inhibit Aβ protein aggregation, and can be applied to the preparation of drugs for neuroprotection.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for preparing Acanthopanax senticosus polysaccharide rich in uronic acid includes the following steps: (1) Add petroleum ether or ethanol to Acanthopanax senticosus root, remove lipids using a Soxhlet extractor, filter, and air dry to obtain defatted Acanthopanax senticosus root; (2) The defatted Acanthopanax senticosus roots obtained in step (1) were subjected to mechanochemical activation pretreatment: placed in a ball mill and heated at a speed of 200-500 r·min - Under certain conditions, the mixture was ball-milled for 30-90 minutes to mechanically disrupt the crystalline regions of the Acanthopanax senticosus cell walls and increase the specific surface area, thereby obtaining activated Acanthopanax senticosus powder. (3) Add the activated Acanthopanax senticosus powder obtained in step (2) to a two-phase system, wherein the two-phase system is composed of polyethylene glycol (PEG) and ammonium sulfate or ammonium phosphate, the molecular weight of PEG is 400-6000, the mass concentration of PEG is 15%-30%, the mass concentration of ammonium sulfate or ammonium phosphate is 10%-25%, and the material-to-liquid ratio is 1:20-50 g·mL. - ¹, After mixing thoroughly, an extraction mixture is obtained; (4) Place the extracted mixture obtained in step (3) into a microwave extraction device for microwave-assisted extraction. The microwave power is 400-800 W, the extraction temperature is 50-70℃, and the extraction time is 10-30 minutes. After extraction, cool and centrifuge to separate the phases. Collect the upper polysaccharide-rich phase to obtain the extract. The microwave-assisted aqueous two-phase extraction efficiently releases acidic polysaccharides while simultaneously removing protein and pigment impurities by utilizing the selective distribution of the aqueous two-phase. (5) Add anhydrous ethanol solution to the extract obtained in step (4) for alcohol precipitation, centrifuge, redissolve the precipitate to obtain the crude polysaccharide solution of Acanthopanax senticosus; (6) Adjust the pH value of the crude polysaccharide solution of Acanthopanax senticosus with dilute hydrochloric acid and dilute sodium hydroxide, and carry out fractional precipitation under pH 3.0, pH 5.0 and pH 7.0 conditions in sequence. Collect the precipitates at pH 5.0 and pH 7.0, discard the precipitate at pH 3.0, and redissolve to obtain pH fractional polysaccharide solution; (7) The pH fractionated polysaccharide solution is passed through a GM-20 membrane with a molecular weight cutoff of 10-50 kDa and separated under operating conditions of 0.20±0.02 MPa and 30±2℃, with the membrane flux decay rate controlled to ≤15%, and the permeate is collected. (8) The permeate was loaded onto a DEAE-650M chromatography column for separation and purification. It was eluted with 0.1 M NaCl, the elution peak was collected, concentrated, dialyzed, and lyophilized to obtain Acanthopanax senticosus polysaccharide rich in uronic acid.

[0010] Furthermore, in step (1), the fat is removed by Soxhlet extractor 2-3 times, each time for 2-3 hours.

[0011] Furthermore, in step (2), the ball-to-material ratio for the ball milling process is 5-20:1, and the grinding balls are made of zirconium oxide or agate.

[0012] Furthermore, the pH of the aqueous two-phase system described in step (3) is 4.0-7.0.

[0013] Furthermore, the centrifugal phase separation conditions described in step (4) are 3000-5000 r·min. - ¹ Centrifuge for 5-10 minutes.

[0014] Furthermore, in step (5), anhydrous ethanol is added until the ethanol volume concentration is 80%; the alcohol precipitation temperature is 4℃ and the time is 12-24h; the centrifugation conditions are: 5000 r·min - ¹, the time is 15-25 minutes.

[0015] Furthermore, the specific operation of pH-fractionated precipitation in step (6) is as follows: the concentration of the crude polysaccharide solution is adjusted to 10 mg·mL. - ¹ First, adjust the pH to 3.0 with 0.1 M hydrochloric acid, let it stand at 4℃ for 2 hours, and centrifuge to remove the precipitate; adjust the pH of the supernatant to 5.0 with 0.1 M sodium hydroxide, let it stand at 4℃ for 2 hours, and centrifuge to collect the precipitate; further adjust the pH of the remaining supernatant to 7.0, let it stand at 4℃ for 2 hours, centrifuge to collect the precipitate, and combine the pH 5.0 and pH 7.0 precipitates.

[0016] Furthermore, the GM-20 membrane separation in step (7) ensures a uniform molecular weight distribution of the obtained polysaccharide, with a weight-average molecular weight of 1.01 × 10⁻⁶. 4 Da, and the uronic acid content remains above 30%.

[0017] Furthermore, the elution procedure for the DEAE-650M chromatography column in step (8) is elution with 0.1 M NaCl.

[0018] The application of a polysaccharide rich in uronic acid in the preparation of a neuroprotective agent is characterized in that the polysaccharide exerts its neuroprotective effect through one or more of the following pathways: reducing the level of reactive oxygen species in CL4176 nematodes, scavenging superoxide anions, inhibiting abnormal Aβ aggregation, delaying Aβ-induced paralysis in nematodes, and enhancing the stress resistance of nematodes.

[0019] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention is the first to combine mechanochemical activation and microwave-assisted aqueous two-phase extraction for the preparation of acidic polysaccharides from Acanthopanax senticosus. Comparative experiments show that the extraction rate of mechanical activation + MATPE (8.56%) is significantly higher than that of unactivated + MATPE (5.23%), proving that the combination of the two produces a synergistic effect.

[0020] (2) This invention combines the above extraction technology with pH-fractionated precipitation (discarding at pH 3.0 and retaining at pH 5.0 and 7.0). Comparative experiments show that after pH-fractionated precipitation, the uronic acid content increased from 27.8% to 38.4%, proving that this strategy has a significant targeted enrichment effect on acidic polysaccharides.

[0021] (3) This invention introduces GM-20 membrane separation technology during the purification process, achieving precise fractionation and homogenization of polysaccharide molecular weight. The resulting ASAP-I HPGPC spectrum shows a single symmetrical peak, with a weight-average molecular weight of 1.01 × 10⁻⁶. 4 Da.

[0022] (4) The ASAP-I prepared in this invention can significantly reduce ROS levels in CL4176 nematodes, scavenge superoxide anions, inhibit abnormal Aβ aggregation, and delay Aβ-induced paralysis in nematodes, of which 50 μg·mL - ¹The ROS inhibition rate in the dose group reached 69%, and the survival rate in the paralysis experiment was 20 percentage points higher than that in the model control group (P<0.01), showing good neuroprotective activity.

[0023] (5) The ASAP-I prepared in this invention can significantly enhance the stress resistance of CL4176 nematodes. In the heat stress experiment, 50 μg·mL - ¹The median survival time in the dosage group was delayed by approximately 8 hours compared to the model control group (P<0.05); in the oxidative stress experiment, 50 μg·mL - ¹The median survival time of the dose group was delayed by about 6 hours compared with the model control group (P<0.05), showing good stress protective activity.

[0024] (6) The preparation method of the present invention is simple to operate, time-saving, efficient and environmentally friendly. The total extraction time is shortened by more than 70% compared with the traditional water extraction method, and the whole process does not involve harmful organic solvents. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0026] Figure 1 The HPGPC spectrum of ASAP-I prepared according to the present invention; Figure 2 This is an HPLC chromatogram of the composition of ASAP-I monosaccharide prepared in this invention; Figure 3 The image shows the HPLC chromatogram of the monosaccharide standard derivative prepared in this invention. Figure 4 The infrared spectrum of ASAP-I prepared according to this invention; Figure 5 This invention relates to the effect of ASAP-I on ROS levels in CL4176 nematodes. Figure 5 Image A is a fluorescence micrograph of nematodes stained with ROS. Figure 5 B is a bar chart showing the relative fluorescence intensity of ROS staining in nematodes; Figure 6 The effect of ASAP-I on superoxide anion levels in CL4176 nematodes according to this invention; Figure 6 Image A is a fluorescence micrograph of nematodes stained with superoxide anion. Figure 6 B is a bar chart showing the relative fluorescence intensity of nematodes stained with superoxide anion; Figure 7 The inhibitory effect of ASAP-I on Aβ aggregation of CL4176 nematodes (thiamine T staining). Figure 7 A is a fluorescence micrograph stained with thiosulfate T in nematodes. Figure 7 B is a bar chart showing the relative fluorescence intensity of nematode thiosulfin T staining; Figure 8 This is a survival curve of Aβ-induced paralysis in CL4176 nematodes induced by ASAP-I of the present invention. Figure 9 This is a bar chart showing the quantitative analysis of PT50 in the Aβ-induced paralysis of CL4176 nematodes by ASAP-I of the present invention. Figure 10 This is a survival curve of ASAP-I in CL4176 nematodes under oxidative stress according to the present invention; Figure 11 This is a survival curve of ASAP-I of the present invention on heat stress in CL4176 nematodes. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1: Mechanochemical activation combined with microwave-assisted aqueous two-phase extraction of Acanthopanax senticosus polysaccharides A method for preparing Acanthopanax senticosus polysaccharide rich in uronic acid includes the following steps: Weigh 2 kg of Acanthopanax senticosus root and place it in a round-bottom flask. Add 30 L of 95% ethanol and defatt it using a Soxhlet extractor for 2 hours. Repeat this process 3 times. After filtration, air-dry the filter residue to obtain defatted Acanthopanax senticosus root.

[0029] The defatted Acanthopanax senticosus roots were placed in a planetary ball mill with a ball-to-material ratio of 10:1. The grinding balls were made of zirconium oxide, and the rotation speed was 300 r·min. - ¹, ball milling for 60 min yielded activated Acanthopanax senticosus powder.

[0030] Preparation of aqueous two-phase system: Weigh 200 g of polyethylene glycol (PEG 4000) and 150 g of ammonium sulfate, dissolve them in water and make up to 1000 mL, adjust the pH to 5.5 to obtain aqueous two-phase solution.

[0031] Take 100 g of activated Acanthopanax senticosus powder and add it to 3000 mL of the above aqueous two-phase solution, with a material-to-liquid ratio of 1:30 g·mL. - ¹, After mixing evenly, the extraction mixture is obtained.

[0032] The extraction mixture was placed in a microwave extraction apparatus, with the microwave power set to 600 W, extraction temperature to 60℃, and extraction time to 20 min. After extraction, the mixture was cooled to room temperature and then extracted at 4000 r·min. - ¹ Centrifuge for 8 minutes to separate the phases, collect the upper phase (PEG phase, polysaccharide enriched phase), and obtain the extract.

[0033] Add 4 times the volume of anhydrous ethanol to the extract until the ethanol concentration reaches 80%, stir well, and let stand overnight at 4°C for alcohol precipitation. Then, precipitate the ethanol at 5000 r·min. - Centrifuge for 20 minutes to redissolve the precipitate and obtain a crude polysaccharide solution of Acanthopanax senticosus.

[0034] The concentration of the crude polysaccharide solution was adjusted to 10 mg·mL. - ¹ First, adjust the pH to 3.0 with 0.1 M hydrochloric acid, let it stand at 4℃ for 2 hours, and centrifuge to remove the precipitate; adjust the pH of the supernatant to 5.0 with 0.1 M sodium hydroxide, let it stand at 4℃ for 2 hours, and centrifuge to collect the precipitate; further adjust the pH of the remaining supernatant to 7.0, let it stand at 4℃ for 2 hours, centrifuge to collect the precipitate, combine the pH 5.0 and pH 7.0 precipitates, and redissolve them with deionized water to obtain a pH-fractionated polysaccharide solution.

[0035] The polysaccharide solution was passed through a GM-20 ceramic membrane and separated under operating conditions of 0.20 MPa and 30°C. The permeate was collected. The membrane flux decay rate was controlled to be ≤15%, and a pulse backwashing method was used to backwash for 15 seconds every 30 minutes to maintain stable membrane flux.

[0036] The permeate was concentrated to 200 mL and loaded onto a DEAE-650M column. Elution was performed with 0.1 M NaCl, and the 0.1 M NaCl elution peak was collected using the phenol-sulfuric acid method. The collected eluent was concentrated under reduced pressure, dialyzed through a 3.5 kDa dialysis bag for 48 h, and then freeze-dried to obtain Acanthopanax senticosus polysaccharide ASAP-I.

[0037] The formula for polysaccharide extraction rate is: Polysaccharide extraction rate (%) = (Mass of polysaccharide sample / Mass of Acanthopanax senticosus raw material) × 100% The uronic acid content was determined using the sulfuric acid carbazole method, expressed as galacturonic acid concentration (mg·mL). - ¹) Plot a standard curve with A as the x-axis and A as the y-axis. Prepare 1 mg·mL - ¹ For ASAP-I solution, perform three parallel experiments as described above, take the average value, measure its absorbance A, and substitute it into the standard curve to calculate the uronic acid content.

[0038] The ASAP-I extraction rate obtained in Example 1 was 8.56%, the uronic acid content was 38.4%, and the obtained polysaccharides had uniform molecular weight with a weight average molecular weight of 1.01 × 10⁻⁶. 4 Da.

[0039] Example 2: Process condition optimization 2.1 Optimization of Mechanochemical Activation Conditions Weigh out three 100 g portions of defatted Acanthopanax senticosus root and, under a ball milling ratio of 10:1, investigate the effects of different ball milling speeds (200 r·min). - ¹、300 r·min - ¹、400 r·min - ¹、500 r·min - ¹) and the effect of different ball milling times (30 min, 60 min, 90 min) on the activation effect were evaluated using the polysaccharide extraction rate under the same aqueous two-phase extraction conditions (PEG 4000 20%, ammonium sulfate 15%, material-liquid ratio 1:30, microwave 600 W, 60℃, 20 min).

[0040] The results showed that the optimal activation conditions were a rotation speed of 300 r·min. - ¹, The extraction time was 60 min, at which point the polysaccharide extraction rate was the highest, reaching 8.56%, which was 63.7% higher than that of the unactivated group (5.23%).

[0041] 2.2 Optimal Composition of the Aqueous Two-Phase System Fixed mechanical activation conditions (300 r·min) -¹, 60 min) and microwave extraction conditions (600 W, 60℃, 20 min) were used to investigate the effects of different PEG molecular weights (400, 1000, 2000, 4000, 6000), different PEG concentrations (15%, 20%, 25%, 30%), and different ammonium sulfate concentrations (10%, 15%, 20%, 25%) on the extraction rate and uronic acid content.

[0042] The results showed that the optimal aqueous two-phase composition was 20% PEG 4000 and 15% ammonium sulfate, at which point the polysaccharide extraction rate was 8.56% and the uronic acid content was 38.4%.

[0043] 2.3 Optimization of Microwave Extraction Conditions Fixed mechanical activation conditions (300 r·min) - ¹, 60 min) and aqueous two-phase compositions (PEG 4000 20%, ammonium sulfate 15%) were used to investigate the effects of different microwave powers (400 W, 500 W, 600 W, 700 W, 800 W), different extraction temperatures (50℃, 55℃, 60℃, 65℃, 70℃), and different extraction times (10 min, 15 min, 20 min, 25 min, 30 min) on the extraction rate and uronic acid content.

[0044] The results showed that the optimal microwave extraction conditions were 600 W power, 60℃ temperature, and 20 min time, under which the polysaccharide extraction rate and uronic acid content reached their highest levels.

[0045] Example 3: Optimization of GM-20 membrane separation parameters pH-fractionated polysaccharide solutions were prepared according to the method in Example 1. The solutions were then passed through a GM-20 ceramic membrane under different operating conditions. The membrane flux decay rate, uronic acid content of the separated polysaccharides, and ROS inhibition activity were compared under different pressures.

[0046] Experimental results showed that when the operating pressure was 0.10 MPa and the temperature was 25℃, the membrane flux decay rate was 12%, the uronic acid content of the obtained polysaccharide was 33.5%, and the ROS inhibition rate was 53%; when the operating pressure was 0.15 MPa and the temperature was 30℃, the membrane flux decay rate was 10%, the uronic acid content of the obtained polysaccharide was 35.1%, and the ROS inhibition rate was 59%; when the operating pressure was 0.20 MPa and the temperature was 30℃, the membrane flux decay rate was 7%, the uronic acid content of the obtained polysaccharide was 38.4%, and the ROS inhibition rate was 69%; when the operating pressure was 0.25 MPa and the temperature was 35℃, the membrane flux decay rate was 14%, the uronic acid content of the obtained polysaccharide was 35.2%, and the ROS inhibition rate was 63%; and when the operating pressure was 0.30 MPa and the temperature was 40℃, the membrane flux decay rate was 20%, the uronic acid content of the obtained polysaccharide was 33.1%, and the ROS inhibition rate was 55%.

[0047] Taking into account membrane flux decay rate, uronic acid content, and ROS inhibition activity, the optimal operating conditions were determined to be a pressure of 0.20 MPa and a temperature of 30℃. Under these conditions, the membrane flux decay rate was the lowest (7%), the uronic acid content was the highest (38.4%), and the ROS inhibition activity was the strongest (69%).

[0048] Furthermore, to highlight the superior performance of the technical solution disclosed in this application compared to the prior art, the following comparative experiments were conducted: Comparative Example 1: Comparison of traditional water extraction methods Acanthopanax senticosus polysaccharide was prepared according to the method of Example 1, except that the traditional water extraction method (material-liquid ratio 1:30, temperature 90℃, extraction for 2 hours) was used instead of mechanical chemical activation-microwave assisted aqueous two-phase extraction. Other steps were the same as in Example 1, and ASAP-IA was prepared.

[0049] Using the method described in Comparative Example 1, the extraction rate of ASAP-IA was 5.52%, and the uronic acid content was 10.8%. This demonstrates that the extraction rate and uronic acid content obtained using the method of this invention are significantly higher than those obtained using the traditional water extraction method.

[0050] Comparative Example 2: Unactivated comparison Acanthopanax senticosus polysaccharide was prepared according to the method of Example 1, except that the mechanical and chemical activation treatment in step (2) was not performed, and the defatted Acanthopanax senticosus root powder was directly extracted by aqueous two-phase extraction. Other steps were the same as in Example 1, and ASAP-IB was prepared.

[0051] Using the method described in Comparative Example 2, the extraction rate of ASAP-IB was 5.23%, and the uronic acid content was 22.6%. This shows that after mechanochemical activation pretreatment, the extraction rate increased from 5.23% to 8.56%, and the uronic acid content increased from 22.6% to 38.4%, demonstrating that mechanochemical activation has a significant destructive effect on the cell wall of Acanthopanax senticosus and can effectively improve the extraction efficiency of acidic polysaccharides.

[0052] Comparative Example 3: Comparison of conventional aqueous two-phase extraction (without microwave) Acanthopanax senticosus polysaccharide was prepared according to the method of Example 1, except that microwave assistance was not used in step (4), and conventional heating and stirring extraction was used (60°C, stirring extraction for 2 hours). Other steps were the same as in Example 1, and ASAP-IC was prepared.

[0053] Using the method described in Comparative Example 3, the extraction rate of ASAP-IC was 6.18%, and the uronic acid content was 28.3%. This shows that microwave-assisted extraction yielded significantly higher extraction rates and uronic acid content than conventional aqueous two-phase extraction.

[0054] Comparative Example 4: pH-fractionated precipitation comparison Acanthopanax senticosus polysaccharide was prepared according to the method of Example 1, except that step (6) of pH fractionation precipitation was not performed, and the other steps were the same as in Example 1, and ASAP-ID was prepared.

[0055] Using the method described in Comparative Example 4, the extraction rate of ASAP-ID was 8.56%, and the uronic acid content was 27.8%. This shows that after pH-fractionated precipitation, the uronic acid content increased from 27.8% to 38.4%, demonstrating that pH-fractionated precipitation has a significant enrichment effect on acidic polysaccharides.

[0056] Experiment 1: Determination of ASAP-I molecular weight and uniformity The molecular weight and homogeneity of ASAP-I obtained in Example 1 were determined by high-performance gel permeation chromatography (HPGPC). Instrument: Agilent 1260II HPLC; Detector: UM4800 evaporative light detector; Column: TSK gel G6000WXL; Column temperature: 40℃; Mobile phase: distilled water; Flow rate: 0.8 mL / min - ¹; Injection volume 20 μL. Prepare ASAP-I at a concentration of 2 mg / mL. - The solution was filtered through a 0.22 µm filter membrane before injection. The detection results are as follows: Figure 1 As shown, the HPGPC spectrum of ASAP-I exhibits a relatively single and symmetrical single peak, and the calculated weight-average molecular weight is 1.01 × 10⁻⁶.4 Da.

[0057] Experiment 2: Determination of ASAP-I monosaccharide composition The monosaccharide composition of ASAP-I was determined using pre-column derivatization-high performance liquid chromatography (PMP). ASAP-I was hydrolyzed with trifluoroacetic acid and then reacted with 0.1 mol·L⁻¹... - The derivatives were derivatized using PMP reagent¹, and separated by a C18 column with UV detection at 250 nm. The monosaccharide composition of ASAP-I was determined by comparing its retention time with that of mixed monosaccharide standard derivatives. The results showed that ASAP-I consists of mannuronic acid, mannose, rhamnose, galacturonic acid, glucose, arabinose, and fucose.

[0058] To further illustrate the application of the uronic acid-rich polysaccharide prepared by this invention in enhancing physical performance, improving stress tolerance, and providing neuroprotection, the inventors conducted the following experiments, the details of which are as follows: Experiment 3: Effects of ASAP-I on ROS levels in CL4176 nematodes A model control group (Mod) and the ASAP-I group prepared in Example 1 were set up. L4-stage CL4176 nematodes that had been synchronized were randomly transferred to the Mod group and groups with different concentrations of ASAP-I (50, 100, 150, 200 μg·mL⁻¹). - ¹) Twenty nematodes were cultured in each dish at 20°C for 48 hours. The nematodes were collected, washed three times with PBS, and then incubated with 10 μM H₂DCF-DA probe at 25°C in the dark for 30 minutes. After washing with PBS, fluorescence was detected using a microplate reader. Compared with the Mod group, administration of ASAP-I prepared in Example 1 resulted in a decrease in ROS levels in the model nematodes. Specifically, ASAP-I (50 μg·mL⁻¹) significantly reduced ROS levels. - The ROS level decreased most significantly in group ¹) (P<0.05), as shown in the following figures. Figure 5 .

[0059] Experiment 4: Effects of ASAP-I on superoxide anion levels in CL4176 nematodes A model control group (Mod) and the ASAP-I group prepared in Example 1 were set up. L4-stage CL4176 nematodes that had been synchronized were randomly transferred to the Mod group and groups with different concentrations of ASAP-I (50, 100, 150, 200 μg·mL⁻¹). -¹) Twenty nematodes were cultured in the culture medium at 20°C for 48 hours. Superoxide anion was specifically detected using a dihydroethidium (DHE) fluorescent probe, and observed and quantitatively analyzed under a fluorescence microscope after staining. Simultaneously, superoxide dismutase (SOD) activity in the nematodes was measured using a superoxide dismutase (SOD) activity assay kit. Compared with the Mod group, administration of ASAP-I prepared in Example 1 resulted in decreased DHE fluorescence intensity and increased SOD activity. Specifically, ASAP-I (50 μg·mL⁻¹) showed the highest concentration of SOD. - Group ¹) showed the most significant effect (P<0.05), as shown in the results below. Figure 6 .

[0060] Experiment 5: Inhibitory effect of ASAP-I on Aβ aggregation of CL4176 nematodes (thiamine T staining) A model control group (Mod) and the ASAP-I group prepared in Example 1 were set up. L4 stage CL4176 nematodes were transferred to the Mod group and groups with different concentrations of ASAP-I (50, 100, 150, 200 μg·mL⁻¹). - ¹) In NGM plates, Aβ expression was induced for 36 hours by incubation at 16°C for 24 hours, followed by warming to 23°C. Nematodes were collected, fixed with 4% paraformaldehyde, and stained with 0.05% thiosulfate T (ThT, dissolved in 50% ethanol) at room temperature for 30 minutes. Observation was performed under a fluorescence microscope. Compared with the Mod group, administration of ASAP-I prepared in Example 1 resulted in a decrease in ThT fluorescence intensity, lighter patch color, and a reduction in the number of patches. Specifically, ASAP-I (50 μg·mL⁻¹) showed the highest ThT fluorescence intensity. - Group ¹) showed the most significant inhibitory effect (P<0.05), as shown in the results below. Figure 7 .

[0061] Experiment 6: The delaying effect of ASAP-I on Aβ-induced paralysis in CL4176 nematodes A model control group (Mod) and the ASAP-I group prepared in Example 1 (50, 100, 150, 200 μg·mL) were set up. - ¹) L4 stage CL4176 nematodes were transferred to NGM plates containing different concentrations of ASAP-I, cultured at 16℃ for 24 hours, and then the temperature was increased to 23℃ to induce Aβ expression. The nematode survival rate was counted after 60 hours.

[0062] The results showed that, compared with the Mod group, all ASAP-I dosage groups could delay Aβ-induced paralysis in nematodes. At 60 hours post-induction, the survival rate in the Mod group was 40%, and the survival rate at 50 μg / mL was [missing data]. - ¹The survival rate in the dosage groups was 60%, at 100, 150, and 200 μg·mL. - ¹The survival rate was 55% in all dosage groups. 50 μg·mL- ¹The dose group showed the best effect, with a 20 percentage point improvement compared to the Mod group (P<0.01), while other dose groups showed a 15 percentage point improvement compared to the Mod group (P<0.05). See results below. Figure 8-9 .

[0063] Experiment 7: Effects of ASAP-I on the stress response of CL4176 nematodes A model control group (Mod) and the ASAP-I group prepared in Example 1 (50, 100, 150, 200 μg·mL) were set up. - ¹). L4 stage CL4176 nematodes treated with ASAP-I for 48 hours were subjected to heat stress and oxidative stress treatment, respectively.

[0064] Heat stress experiment: Nematodes were transferred to a 35℃ incubator, and survival was recorded every 2 hours. Results showed that, compared with the Mod group, ASAP-I pretreatment significantly enhanced the heat stress tolerance of nematodes, with 50 μg·mL⁻¹ being the most effective. - ¹The dosage group showed the best effect. After 14 hours of heat stress treatment, the survival rate in the Mod group was 30%, while that in the 50 μg·mL group was... - ¹The survival rate in the dosage group was 66.67%; after 20 hours of heat stress treatment, the survival rate in the Mod group decreased to 3.33%, while the survival rate in the 50 μg·mL group was higher. - ¹36.67% of the nematodes in the dosage group remained alive. 50 μg·mL - ¹The median survival time in the dose group was delayed by approximately 8 hours compared to the Mod group (P<0.05).

[0065] Oxidative stress experiment: Nematodes were transferred to a culture medium containing an oxidative inducer, and survival was assessed every 2 hours. Results showed that, compared to the Mod group, ASAP-I pretreatment significantly enhanced the nematode's tolerance to oxidative stress, with 50 μg / mL... - ¹The dosage group showed the best results. At 22 hours post-induction, the survival rate in the Mod group was 0%, while the 50 μg / mL group... - ¹The survival rate in the dose group was 20%; 24 hours after induction, 50 μg·mL - ¹13.33% of the nematodes in the dosage group remained alive. 50 μg·mL - ¹The median survival time in the dose group was delayed by approximately 6 hours compared to the Mod group (P<0.05).

[0066] The above results indicate that ASAP-I can significantly enhance the stress resistance of CL4176 nematodes. See the results below. Figure 10-11 .

[0067] In summary, the experimental results show that the ASAP-I uronic acid prepared in this invention has high content and uniform molecular weight, and can significantly reduce ROS levels in the CL4176 nematode model, scavenge superoxide anions, inhibit Aβ aggregation, and enhance the motility and stress resistance of the nematode model, demonstrating good neuroprotective potential.

[0068] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing Acanthopanax senticosus polysaccharide rich in uronic acid, characterized in that, Includes the following steps: (1) Add petroleum ether or ethanol to Acanthopanax senticosus root, remove lipids using a Soxhlet extractor, filter, and air dry to obtain defatted Acanthopanax senticosus root; (2) The defatted Acanthopanax senticosus roots obtained in step (1) were subjected to mechanochemical activation pretreatment: placed in a ball mill and heated at a speed of 200-500 r·min - Under certain conditions, the mixture was ball-milled for 30-90 minutes to mechanically disrupt the crystalline regions of the Acanthopanax senticosus cell walls and increase the specific surface area, thereby obtaining activated Acanthopanax senticosus powder. (3) Add the activated Acanthopanax senticosus powder obtained in step (2) to a two-phase system, wherein the two-phase system is composed of polyethylene glycol (PEG) and ammonium sulfate or ammonium phosphate, the molecular weight of PEG is 400-6000, the mass concentration of PEG is 15%-30%, the mass concentration of ammonium sulfate or ammonium phosphate is 10%-25%, and the material-to-liquid ratio is 1:20-50 g·mL. - ¹, After mixing thoroughly, an extraction mixture is obtained; (4) Place the extracted mixture obtained in step (3) into a microwave extraction device for microwave-assisted extraction. The microwave power is 400-800 W, the extraction temperature is 50-70℃, and the extraction time is 10-30 minutes. After extraction, cool and centrifuge to separate the phases. Collect the upper polysaccharide-rich phase to obtain the extract. The microwave-assisted aqueous two-phase extraction efficiently releases acidic polysaccharides while simultaneously removing protein and pigment impurities by utilizing the selective distribution of the aqueous two-phase. (5) Add anhydrous ethanol solution to the extract obtained in step (4) for alcohol precipitation, centrifuge, redissolve the precipitate to obtain the crude polysaccharide solution of Acanthopanax senticosus; (6) Adjust the pH value of the crude polysaccharide solution of Acanthopanax senticosus with dilute hydrochloric acid and dilute sodium hydroxide, and carry out fractional precipitation under pH 3.0, pH 5.0 and pH 7.0 conditions in sequence. Collect the precipitates at pH 5.0 and pH 7.0, discard the precipitate at pH 3.0, and redissolve to obtain pH fractional polysaccharide solution; (7) The pH fractionated polysaccharide solution is passed through a GM-20 membrane with a molecular weight cutoff of 10-50 kDa and separated under operating conditions of 0.20±0.02 MPa and 30±2℃, with the membrane flux decay rate controlled to ≤15%, and the permeate is collected. (8) The permeate was loaded onto a DEAE-650M chromatography column for separation and purification. It was eluted with 0.1 M NaCl, the elution peak was collected, concentrated, dialyzed, and lyophilized to obtain Acanthopanax senticosus polysaccharide rich in uronic acid.

2. The preparation method according to claim 1, characterized in that, In step (1), the fat is removed by Soxhlet extractor 2-3 times, each time for 2-3 hours.

3. The preparation method according to claim 1, characterized in that, The ball-to-material ratio for the ball milling process in step (2) is 5-20:1, and the grinding balls are made of zirconium oxide or agate.

4. The preparation method according to claim 1, characterized in that, The pH of the aqueous two-phase system described in step (3) is 4.0-7.

0.

5. The preparation method according to claim 1, characterized in that, The centrifugal phase separation conditions described in step (4) are 3000-5000 r·min. - ¹ Centrifuge for 5-10 minutes.

6. The preparation method according to claim 1, characterized in that, In step (5), anhydrous ethanol is added until the ethanol volume concentration is 80%; the alcohol precipitation temperature is 4℃ and the time is 12-24h; the centrifugation conditions are: 5000 r·min - ¹, the time is 15-25 minutes.

7. The preparation method according to claim 1, characterized in that, The specific operation of pH fractionation precipitation in step (6) is as follows: adjust the concentration of the crude polysaccharide solution to 10 mg·mL. - ¹ First, adjust the pH to 3.0 with 0.1 M hydrochloric acid, let it stand at 4℃ for 2 hours, and then centrifuge to remove the precipitate; The supernatant was adjusted to pH 5.0 with 0.1 M sodium hydroxide, and the precipitate was collected by centrifugation after standing at 4°C for 2 hours. The remaining supernatant was further adjusted to pH 7.0, and the precipitate was collected by centrifugation after standing at 4°C for 2 hours. The pH 5.0 and pH 7.0 precipitates were combined.

8. The preparation method according to claim 1, characterized in that, The GM-20 membrane separation in step (7) ensures that the obtained polysaccharide has a uniform molecular weight distribution, with a weight-average molecular weight of 1.01 × 10⁻⁶. 4 Da, and the uronic acid content remains above 30%.

9. The preparation method according to claim 1, characterized in that, The elution procedure for the DEAE-650M chromatography column in step (8) is to elute with 0.1 M NaCl.

10. The application of a uronic acid-rich Acanthopanax senticosus polysaccharide prepared by the method according to any one of claims 1-9 in the preparation of a neuroprotective agent, characterized in that, The polysaccharide exerts its neuroprotective effect through one or more of the following pathways: reducing reactive oxygen species levels in CL4176 nematodes, scavenging superoxide anions, inhibiting abnormal Aβ aggregation, delaying Aβ-induced paralysis in nematodes, and enhancing the stress response of nematodes.

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