Acanthopanax senticosus-proteoglycan protein compound as well as preparation method and application thereof

Abstract

The invention discloses an acanthopanax senticosus-polysaccharide protein compound as well as a preparation method and application thereof, and belongs to the technical field of biology. According to the preparation method of the acanthopanax senticosus-proteoglycan protein compound, acanthopanax senticosus is taken as a raw material, and the acanthopanax senticosus-proteoglycan protein compound is extracted by adopting a microwave-enzyme synergistic extraction method. The acanthopanax senticosus-polysaccharide protein compound disclosed by the invention can inhibit Abeta-induced apoptosis in CL4176 caenorhabditis elegans, and shows remarkable effects in the aspects of prolonging the service life, reducing the paralysis rate, improving the athletic ability, enhancing antioxidant defense and the like.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to an Acanthopanax senticosus polysaccharide-protein complex, its preparation method, and its application in the preparation of a drug for inhibiting apoptosis in CL4176 Caenorhabditis elegans cells. Background Technology

[0002] Apoptosis, a key physiological process for maintaining homeostasis in organisms, plays a crucial role in neurodegenerative changes, oxidative stress damage, and age-related diseases. Abnormally elevated apoptosis levels can lead to decreased tissue function, impaired motor function, or premature nerve cell death; conversely, insufficient apoptosis inhibition can result in abnormal cell proliferation. Therefore, developing natural macromolecules that can regulate apoptosis, improve the body's antioxidant capacity, and delay functional decline is an important direction in current biomedical and functional resource development.

[0003] Plant polysaccharides have become a key focus of natural product research due to their wide availability, high biocompatibility, and diverse biological activities, including immunomodulation and antioxidant effects. However, their independent use still has limitations: polysaccharides have large molecular weights and complex structures, leading to instability in components due to different extraction processes; furthermore, polysaccharides are often accompanied by impurities such as proteins, making purification difficult and resulting in unsatisfactory bioavailability. Recent studies have shown that when polysaccharides form stable complexes with proteins, their physicochemical properties and biological activities can be simultaneously improved, such as enhancing free radical scavenging ability, increasing stability, and improving in vivo absorption. Therefore, developing plant polysaccharide-protein complexes has become an important strategy for enhancing their activity.

[0004] Acanthopanax senticosus is a traditional medicinal plant belonging to the Araliaceae family, containing various active substances such as polysaccharides, saponins, and flavonoids. Among them, Acanthopanax senticosus polysaccharides have shown good antioxidant, anti-fatigue, and immunomodulatory effects, and can participate in cellular stress responses and delay the damage process. However, natural Acanthopanax senticosus polysaccharides suffer from problems such as complex structure, many impurities, and low extraction efficiency, which are not conducive to their use as stable macromolecular active substances in pharmacological research or product development.

[0005] Furthermore, existing polysaccharide extraction techniques, such as hot water extraction and ultrasonic extraction, suffer from drawbacks such as low efficiency, insufficient cell wall disruption, and loss of bioactivity; while simple enzyme extraction is time-consuming and difficult to balance efficiency and activity preservation. Microwave-enzyme synergistic extraction technology is a composite technology that combines the advantages of microwave cell disruption with enzyme-catalyzed specific degradation, which can significantly improve extraction efficiency and improve the structural integrity of polysaccharides, providing a better solution for the preparation of polysaccharide-protein complexes.

[0006] However, there is currently no systematic literature reporting the acquisition of stable polysaccharide-protein complexes from Acanthopanax senticosus using a microwave-enzyme synergistic approach, nor evaluating their comprehensive biological activities in prolonging lifespan, improving motor function, enhancing antioxidant capacity, and regulating apoptosis. Particularly in the area of ​​neurotoxicity models, there is a lack of systematic research on the regulation of Aβ-induced apoptosis and stress responses by these complexes.

[0007] Therefore, it is urgent to develop an efficient, mild, and stable method for preparing Acanthopanax senticosus polysaccharide-protein complex to obtain a structurally intact and biologically stable macromolecule and to verify its effects on prolonging lifespan, reducing paralysis rate, improving motor ability, enhancing antioxidant capacity, and inhibiting cell apoptosis in the Caenorhabditis elegans (CL4176) model. Summary of the Invention

[0008] In view of this, the present invention provides an Acanthopanax senticosus-polysaccharide-protein complex, its preparation method, and its application. Using Acanthopanax senticosus as raw material, the Acanthopanax senticosus-polysaccharide-protein complex ASPS-Protein Complex is extracted using a microwave-enzyme synergistic extraction method. The ASPS-Protein Complex of the present invention can inhibit apoptosis in CL4176 Caenorhabditis elegans cells, and the effect is more significant after using the ASPS-Protein Complex compared to the natural state.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing an Acanthopanax senticosus-polysaccharide-protein complex includes the following steps: (1) Add petroleum ether to the powder of Acanthopanax senticosus to defatt the powder, filter and dry to obtain defatted Acanthopanax senticosus; add citrate-sodium citrate buffer to the defatted Acanthopanax senticosus, then add neutral cellulase and place it in a water bath shaker for pre-enzymatic hydrolysis to ensure full contact. (2) Take the pre-enzymatic hydrolysis system obtained in step (1) and transfer it to the polytetrafluoroethylene reaction vessel of the microwave extractor for microwave extraction. (3) After the microwave extraction in step (2) is completed, the reaction vessel is placed in a boiling water bath for heating and then cooled. After centrifugation, the supernatant I and the residue are collected. The residue is extracted once again according to the above steps to obtain supernatant II. The two supernatants are combined to obtain the crude extract of Acanthopanax senticosus. (4) The crude extract of Acanthopanax senticosus obtained in step (3) was eluted with macroporous adsorption resin and purified with dialysis bag and then freeze-dried to obtain Acanthopanax senticosus-polysaccharide-protein complex ASPS-Protein Complex.

[0010] Microwave-assisted enzymatic methods can save extraction time, reduce energy consumption, and improve extraction rates. Microwave-enzyme synergistic extraction is a novel extraction technology that combines the efficient cell-wall breaking ability of microwaves with the specific degradation effect of enzymes. Through the synergistic effect of "physical field enhancement + biocatalysis," it solves the problems of incomplete action of microwave extraction on dense cell walls and the long extraction time of single enzyme extraction, achieving a higher extraction efficiency, optimized balance between activity retention and process economy.

[0011] Furthermore, in step (1), the volume ratio of Acanthopanax senticosus to petroleum ether is 1:5 g / mL; the boiling range of the petroleum ether is 60-90℃.

[0012] Further, in step (1), the ratio of defatted Acanthopanax senticosus to citrate-sodium citrate buffer is (1:10~20) g / mL; the concentration of citrate-sodium citrate buffer is 0.1 mol / L and the pH is 4.5.

[0013] Furthermore, the temperature of the water bath shaker in step (1) is 40-50℃.

[0014] Furthermore, in step (2), the amount of neutral cellulase added is 1.0%, the enzymatic hydrolysis time is 30 min, and the enzyme activity of the neutral cellulase is 10,000 U / g.

[0015] Furthermore, in step (2), the microwave power is 250-750W, the microwave temperature is 45℃, the microwave time is 10-30min, and the pressure is 0.1 MPa.

[0016] Furthermore, the heating time in the boiling water bath in step (3) is 10 minutes.

[0017] Further, in step (4), the crude extract of Acanthopanax senticosus is passed through D101 macroporous resin, dialyzed in a 3500Da dialysis bag for 2-3 days, and then freeze-dried to obtain the Acanthopanax senticosus-polysaccharide-protein complex ASPS-Protein Complex.

[0018] Furthermore, the Acanthopanax senticosus-polysaccharide protein complex prepared by the method is obtained.

[0019] Furthermore, the application of the Acanthopanax senticosus-polysaccharide protein complex in the preparation of drugs that prolong the lifespan of nematodes, reduce the paralysis rate of nematodes, improve the motility of nematodes, enhance the stress resistance of nematodes, and / or inhibit apoptosis in CL4176 Caenorhabditis elegans cells.

[0020] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a Acanthopanax senticosus-polysaccharide-protein complex, its preparation method and application, which has the following beneficial effects: (1) The present invention uses a novel microwave-assisted enzymatic method to extract Acanthopanax senticosus-polysaccharide protein complex, which can not only solve the shortcomings of low polysaccharide extraction efficiency and poor purity due to the hard cell wall of Acanthopanax senticosus, but also retain the biological activity of macromolecular substances to a greater extent.

[0021] (2) This invention describes that if it is necessary to quickly obtain highly active ASPS-Protein Complex for pharmacological experiments (such as immune regulation and anti-fatigue research), efficient extraction can be achieved by using a small microwave extractor (within 500W) and a compound enzyme.

[0022] (3) The present invention uses CL4176 Caenorhabditis elegans as a model and applies the ASPS-Protein Complex prepared in the present invention to administer the drug. The results show that the macromolecules of the present invention can inhibit apoptosis of CL4176 Caenorhabditis elegans cells. Compared with the natural state, the effect of using ASPS-Protein Complex is more significant. Attached Figure Description

[0023] 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.

[0024] Figure 1 The graph shows the effect of different samples (sample 1 to sample D) on the paralysis process of Caenorhabditis elegans CL4176.

[0025] Figure 2 A bar chart showing the bowing frequency of CL4176 nematodes for different samples (sample 1 to sample D).

[0026] Figure 3 The survival rate curves of CL4176 nematodes in the Con group and the sample 1-3 treatment groups are shown under Juglone acute oxidative stress.

[0027] Figure 4 Bar chart showing the SOD activity, CAT activity, and MDA content in the homogenate of CL4176 nematodes in the Con group and treatment groups 1-3.

[0028] Figure 5 A bar chart showing the effect of stress-related genes (jnk-1, daf-16, sod-3, atm-1) expression levels on the Con group and the treatment groups 1-3 of Caenorhabditis elegans CL4176. Detailed Implementation

[0029] 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.

[0030] Example 1 A method for preparing an Acanthopanax senticosus-polysaccharide-protein complex includes the following steps: Weigh 1,000 kg of Acanthopanax senticosus powder and add petroleum ether (boiling range 60-90 ℃) at a material-to-liquid ratio of 1:5 g / mL. Reflux twice in a 60 ℃ constant temperature water bath for 2 h each time to defatt the powder. After defatting, filter while hot and collect the solid residue. Dry the solid residue in a 50–60 ℃ forced-air drying oven until no solvent odor remains to obtain defatted Acanthopanax senticosus. Add the defatted Acanthopanax senticosus powder to an Erlenmeyer flask and add approximately 15 L of 0.1 mol / L citrate-sodium citrate buffer (pH 4.5) at a material-to-liquid ratio of 1:15 g / mL. Then add 10.0 g of neutral cellulase (10,000 U / g) at 1.0% of the weight of the Acanthopanax senticosus powder. Gently shake to ensure the enzyme is fully in contact with the Acanthopanax senticosus powder. Place the Erlenmeyer flask in a 45 ℃ constant temperature water bath shaker for pre-enzymatic hydrolysis for 30 min. The pre-enzymatically digested system was transferred in batches to the polytetrafluoroethylene reaction vessel of a microwave extractor. The parameters were set as follows: 500 W, 45 ℃, 20 min, 0.1 MPa. After extraction, the reaction vessel was immediately placed in a boiling water bath for 10 min, cooled to room temperature, and centrifuged at 8000 rpm for 15 min. The supernatant was collected. The residue after centrifugation was extracted once more using the same steps (from the addition of citrate-sodium citrate buffer to the end), and the supernatants from both extractions were combined.

[0031] Pretreated D101 macroporous resin was wet-packed into a glass chromatography column (column dimensions: Φ8.5 cm × 30 cm, bed volume approximately 1.7 L). The column bed was washed with distilled water until the eluent was clear, with a flow rate controlled at 1-2 BV / h. The combined supernatant was filtered through a 0.45 μm filter membrane and loaded onto the resin column at a flow rate of 1 BV / h. After loading, the column was eluted with distilled water at a flow rate of 2 BV / h, and the eluent was collected. After elution, the resin was eluted with 95% ethanol at a flow rate of 2 BV / h until the eluent was colorless, and then washed with distilled water until no alcohol odor was detected.

[0032] Cut a 3500 Da dialysis bag into approximately 20 cm segments, boil them in distilled water at 50 °C for 10 min, then soak them in 0.05 mol / L EDTA solution for 30 min, and finally rinse with distilled water until neutral. Place the eluent into the pretreated dialysis bag, tie both ends tightly, and place it in a beaker containing sufficient distilled water. Dialyze with magnetic stirring at 4 °C, changing the distilled water every 4 h for 2-3 days until no Cl- is detected in the external solution using AgNO3 solution. - .

[0033] The dialyzed solution was transferred to a rotary evaporator and concentrated under reduced pressure at 45 °C to 1 / 5 of its original volume. The concentrate was then poured into a lyophilization bottle and placed in a freeze dryer. It was pre-frozen at -80 °C for 4 h and then vacuum dried for 24-48 h until a loose white or pale yellow ASPS-Protein Complex-1 powder was obtained.

[0034] The protein content of ASPS-Protein Complex-1 was determined using the BCA protein quantification method. The results showed that the protein content in the complex was approximately 15.2% (w / w). Combined with the polysaccharide content determined by the phenol-sulfuric acid method, the polysaccharide content was approximately 74.47% (w / w), and the total extraction rate of the complex was approximately 8.51%. These results indicate that this complex is a polysaccharide-protein hybrid, retaining the protein components and facilitating biological activity. Furthermore, the extraction efficiency is suitable for both laboratory and pilot-scale production.

[0035] Example 2 Weigh 1,000 kg of Acanthopanax senticosus powder and add petroleum ether (boiling range 60–90 ℃) at a material-to-liquid ratio of 1:5 g / mL. Reflux twice in a 60 ℃ constant temperature water bath for 2 h each time to defatt the powder. After defatting, filter while hot and collect the solid residue. Dry the solid residue in a 50–60 ℃ forced-air drying oven until no solvent odor remains to obtain defatted Acanthopanax senticosus. Add the defatted Acanthopanax senticosus powder to an Erlenmeyer flask and add approximately 10 L of 0.1 mol / L citrate-sodium citrate buffer (pH 4.5) at a material-to-liquid ratio of 1:10 g / mL. Then add 10.0 g of neutral cellulase (10,000 U / g) at 1.0% of the weight of the Acanthopanax senticosus powder. Gently shake to ensure the enzyme is fully in contact with the Acanthopanax senticosus powder. Place the Erlenmeyer flask in a 40 ℃ constant temperature water bath shaker for pre-enzymatic hydrolysis for 30 min. The pre-enzymatically digested system was transferred in batches to the polytetrafluoroethylene reaction vessel of a microwave extractor. The parameters were set as follows: 250 W, 45 ℃, 10 min, 0.1 MPa. After extraction, the reaction vessel was immediately placed in a boiling water bath for 10 min, cooled to room temperature, and centrifuged at 8000 rpm for 15 min. The supernatant was collected. The residue after centrifugation was extracted once more using the same steps (from the addition of citrate-sodium citrate buffer to the end), and the supernatants from both extractions were combined.

[0036] Pretreated D101 macroporous resin was wet-packed into a glass chromatography column (column dimensions: Φ8.5 cm × 30 cm, bed volume approximately 1.7 L). The column bed was washed with distilled water until the eluent was clear, with a flow rate controlled at 1–2 BV / h. The combined supernatant was filtered through a 0.45 μm filter membrane and loaded onto the resin column at a flow rate of 1 BV / h. After loading, the column was eluted with distilled water at a flow rate of 2 BV / h, and the eluent was collected. After elution, the resin was eluted with 95% ethanol at a flow rate of 2 BV / h until the eluent was colorless, and then washed with distilled water until no alcohol odor was detected.

[0037] Cut the 3500 Da dialysis bag into approximately 20 cm segments, boil them in distilled water at 50 °C for 10 min, then soak them in 0.05 mol / L EDTA solution for 30 min, and finally rinse with distilled water until neutral. Place the eluent into the pretreated dialysis bag, tie both ends tightly, and place it in a beaker containing sufficient distilled water. Dialyze with magnetic stirring at 4 °C, changing the distilled water every 4 h for 2–3 days until no Cl- is detected in the external solution using AgNO3 solution. - .

[0038] The dialyzed solution was transferred to a rotary evaporator and concentrated under reduced pressure at 45 °C to 1 / 5 of its original volume. The concentrate was poured into a lyophilization bottle and placed in a freeze dryer. It was pre-frozen at -80 °C for 4 h and then vacuum dried for 24-48 h until a loose white or pale yellow ASPS-Protein Complex-2 powder was obtained.

[0039] The protein content of ASPS-Protein Complex-2 was determined using the BCA protein quantification method. The results showed that the protein content in the complex was approximately 12.1% (w / w). Combined with the polysaccharide content determined by the phenol-sulfuric acid method, the content was approximately 69.47% (w / w), and the total extraction rate of the complex was approximately 6.91%.

[0040] Example 3 Weigh 1,000 kg of Acanthopanax senticosus powder and add petroleum ether (boiling range 60–90 ℃) at a material-to-liquid ratio of 1:5 g / mL. Reflux twice in a 60 ℃ constant temperature water bath for 2 h each time to defatted the powder. After defatting, filter while hot and collect the solid residue. Dry the solid residue in a 50–60 ℃ forced-air drying oven until no solvent odor remains to obtain defatted Acanthopanax senticosus. Add the defatted Acanthopanax senticosus powder to an Erlenmeyer flask and add approximately 20 L of 0.1 mol / L citrate-sodium citrate buffer (pH 4.5) at a material-to-liquid ratio of 1:20 g / mL. Then add 10.0 g of neutral cellulase (10,000 U / g) at 1.0% of the weight of the Acanthopanax senticosus powder. Gently shake to ensure the enzyme is fully in contact with the Acanthopanax senticosus powder. Place the Erlenmeyer flask in a 50 ℃ constant temperature water bath shaker for pre-enzymatic hydrolysis for 30 min. The pre-enzymatically digested system was transferred in batches to the polytetrafluoroethylene reaction vessel of a microwave extractor. The parameters were set as follows: 750 W, 45 ℃, 30 min, 0.1 MPa. After extraction, the reaction vessel was immediately placed in a boiling water bath for 10 min, cooled to room temperature, and centrifuged at 8000 rpm for 15 min. The supernatant was collected. The residue after centrifugation was extracted once more using the same steps (from the addition of citrate-sodium citrate buffer to the end), and the supernatants from both extractions were combined.

[0041] Pretreated D101 macroporous resin was wet-packed into a glass chromatography column (column dimensions: Φ8.5 cm × 30 cm, bed volume approximately 1.7 L). The column bed was washed with distilled water until the eluent was clear, with a flow rate controlled at 1–2 BV / h. The combined supernatant was filtered through a 0.45 μm filter membrane and loaded onto the resin column at a flow rate of 1 BV / h. After loading, the column was eluted with distilled water at a flow rate of 2 BV / h, and the eluent was collected. After elution, the resin was eluted with 95% ethanol at a flow rate of 2 BV / h until the eluent was colorless, and then washed with distilled water until no alcohol odor was detected.

[0042] Cut the 3500 Da dialysis bag into approximately 20 cm segments, boil them in distilled water at 50 °C for 10 min, then soak them in 0.05 mol / L EDTA solution for 30 min, and finally rinse with distilled water until neutral. Place the eluent into the pretreated dialysis bag, tie both ends tightly, and place it in a beaker containing sufficient distilled water. Dialyze with magnetic stirring at 4 °C, changing the distilled water every 4 h for 2–3 days until no Cl- is detected in the external solution using AgNO3 solution. - .

[0043] The dialyzed solution was transferred to a rotary evaporator and concentrated under reduced pressure at 45 °C to 1 / 5 of its original volume. The concentrate was then poured into a lyophilization bottle and placed in a freeze dryer. It was pre-frozen at -80 °C for 4 h and then vacuum dried for 24-48 h until a loose white or pale yellow ASPS-Protein Complex-3 powder was obtained.

[0044] The protein content of ASPS-Protein Complex-3 was determined using the BCA protein quantification method. The results showed that the protein content in the complex was approximately 13.9% (w / w). Combined with the phenol-sulfuric acid method, the polysaccharide content was approximately 65.47% (w / w), and the total extraction rate of the complex was approximately 7.36%.

[0045] Comparative Example 1 The Acanthopanax senticosus-polysaccharide-protein complex was prepared according to the method of Example 1, except that acidic cellulase (10,000 U / g) was added during the extraction process. Other steps were the same as in Example 1 to prepare the Acanthopanax senticosus-polysaccharide-protein complex ASPS-Protein Complex-A.

[0046] Using the method of Comparative Example 1, the total extraction rate of ASPS-Protein Complex-A was 5.74%, the sugar content was 51.63%, and the protein content was approximately 10.43% (w / w).

[0047] Comparative Example 2 The Acanthopanax senticosus-polysaccharide-protein complex was prepared according to the method of Example 1, except that when the pre-enzymatically hydrolyzed system was transferred in batches to the polytetrafluoroethylene reaction vessel of the microwave extractor during the extraction process, the temperature of the reaction vessel system was set to 55°C. Other steps were the same as in Example 1 to prepare the Acanthopanax senticosus-polysaccharide-protein complex ASPS-Protein Complex-B.

[0048] Using the method of Comparative Example 2, the total extraction rate of ASPS-Protein Complex-B was 8.43%, the sugar content was 53.98%, and the protein content was approximately 6.79% (w / w).

[0049] Comparative Example 3 The Acanthopanax senticosus-polysaccharide-protein complex was prepared according to the method of Example 1, except that the Acanthopanax senticosus-polysaccharide-protein complex ASPS-Protein Complex was prepared by traditional hot water extraction and ultrasonic extraction respectively. Other steps were the same as in Example 1, and ASPS-Protein Complex-C and ASPS-Protein Complex-D were obtained.

[0050] Traditional hot water method: Weigh 100.0g of Acanthopanax senticosus powder, add petroleum ether solution at a ratio of 1:5g / mL, reflux and defatt the solution in a 60℃ constant temperature water bath, then add distilled water at a ratio of 1:20 and soak at 95℃ for 2-4 hours; filter the extract while hot to remove residue; concentrate the filtrate to 1 / 10 of the original volume, add 4 times the volume of 95% ethanol, and let stand at 4℃ for 24 hours to precipitate the complex; collect the precipitate by centrifugation at 3500r / min for 15min, wash 3 times with anhydrous ethanol, and dry to obtain crude ASPS-Protein Complex. Subsequent purification (eluting with macroporous adsorption resin and removing impurities with a dialysis bag, followed by freeze drying) is the same as in Example 1.

[0051] Ultrasonic extraction method: Weigh 100.0g of Acanthopanax senticosus powder, add petroleum ether solution at a ratio of 1:5g / mL, reflux and defatt the mixture in a 60℃ constant temperature water bath, then add distilled water at a ratio of 1:20 and stir to completely wet the powder. Transfer the mixture to a stoppered conical flask and place it in an ultrasonic extractor. Set the power to 400W and the temperature to 60℃, and perform intermittent ultrasonication three times, each time for 15 minutes, with a 5-minute interval, and keep the entire process sealed. After ultrasonication, filter the mixture through double-layer gauze and collect the filtrate. Add distilled water to the residue at the same ratio and repeat ultrasonication for 30 minutes, then combine the two filtrates. Concentrate the filtrate to 1 / 10 of its original volume, add 4 times the volume of 95% ethanol, and let it stand at 4℃ for 24 hours to precipitate the complex. Centrifuge at 3500r / min for 15 minutes, collect the precipitate, wash it three times with anhydrous ethanol, and dry it to obtain crude ASPS-Protein Complex. Subsequent purification (eluting with macroporous adsorption resin, removing impurities with a dialysis bag, and then freeze-drying) is the same as in Example 1.

[0052] Using the method of Comparative Example 3, the total extraction rate of ASPS-Protein Complex-C was 5.81%, the sugar content was 49.16%, and the protein content was approximately 11.26%; the extraction rate of ASPS-Protein Complex-D was 5.96%, the sugar content was 52.38%, and the protein content was approximately 9.67%.

[0053] Therefore, it can be concluded that the extraction rate and sugar content of Acanthopanax senticosus polysaccharide in microwave-assisted enzymatic extraction are significantly higher than those in traditional hot water extraction and ultrasonic extraction. Microwave-enzyme synergistic extraction is a novel extraction technology that combines the efficient cell wall breaking ability of microwaves with the specific degradation effect of enzymes. Through the synergistic effect of "physical field enhancement + biocatalysis", it solves the problems of incomplete action of microwave extraction on dense cell walls and long extraction time of single enzymes, and achieves an optimized balance between ASPS extraction efficiency, activity retention and process economy.

[0054] After obtaining the Acanthopanax senticosus polysaccharide-protein complex, the present invention conducted the following systematic experimental study to investigate its regulatory effects on the lifespan, stress resistance, and apoptosis process of Caenorhabditis elegans type CL4176.

[0055] Experimental Example 1 Lifespan Experiment: A blank control group (Con) and the ASPS-Protein Complex prepared in Examples 1-3 were set up, corresponding to samples 1, 2, and 3, respectively. Comparative Examples 1-3 also included ASPS-Protein Complex, corresponding to samples A, B, C, and D, respectively. Three dose gradients (0.75, 1.00, and 1.25 mg / mL) were set up for each sample. CL4176 nematodes synchronized to the L3 stage were inoculated into NGM solid medium containing different samples and corresponding doses, with 30 nematodes per medium, and cultured at 16 °C. To avoid interference from egg and larval growth on the counting results, 5-fluorouracil (5-FU, final concentration 50 μM) was added to the NGM to inhibit nematode oviposition. Every two days, surviving nematodes were transferred to a new corresponding NGM medium until all nematodes died. The number of surviving nematodes in each group was recorded daily, and the average and maximum lifespans were calculated to screen for the optimal drug concentration. The results are shown in Table 1.

[0056] Table 1

[0057] Note: Different lowercase letters (a–f) after the data in the same column indicate statistically significant differences (p<0.05), while the same letter indicates no significant differences (p>0.05).

[0058] Table 1 shows that all seven samples exhibited varying degrees of lifespan extension. Sample 1 showed the most significant effect, followed by Sample 2, with Sample 3 showing moderate significance. Samples A and B showed slight lifespan extension, while there was no statistically significant difference between Samples C and D. Overall, the results indicate that Sample 1 had the best lifespan-promoting effect at 1.0 mg / mL, and this dosage was used in subsequent experiments.

[0059] Experimental Example 2 Paralysis experiment: A blank group (Con) and medium-dose groups (1.00 mg / mL) of samples 1, 2, 3, A, B, C, and D were set up. CL4176 nematodes synchronized to stage L3 were inoculated into NGM solid medium containing different samples, with 30 nematodes per medium. After incubation at 16 °C for 36 h, the temperature was increased to 25 °C for another 28 h. The paralysis status of the nematodes was observed and recorded under a microscope every 2 h. Paralysis was defined as no response to touching the nematode's body or head, or only slight head movement without body movement. Each group underwent three parallel experiments, and the cumulative paralysis rate of the nematodes at each time point was calculated. The results are shown in Table 2. Figure 1 .

[0060] Table 2. Effects of different samples on the paralysis process of CL4176 nematodes (n = 30, statistics every 2 h); cumulative paralysis rate (%)

[0061] The results showed that, compared with the Con group, samples 1, 2, 3 and A-D could reduce the paralysis rate of CL4176 nematodes and delay the onset of paralysis to varying degrees. The effects of samples 1, 2 and 3 were more significant, while the improvement of samples A-D was relatively smaller.

[0062] Experimental Example 3 Motility test: To evaluate the effect of each sample on the motility of CL4176 Caenorhabditis elegans, nematodes synchronized to the L3 stage were inoculated into NGM plates of the Con group and samples 1 to D (concentration of 1.0 mg·mL⁻¹). -1 The nematodes were cultured at 16 °C for 36 h, and then the temperature was increased to 25 °C to induce the Aβ phenotype. At 48 h, 10 nematodes were randomly selected and placed in a sterile M9 buffer drop, and the spontaneous movement of the nematodes was observed under an inverted microscope.

[0063] One complete bending of the nematode's body within 30 seconds (leftward + rightward counts as one bending) was defined as one cycle. The bending frequency of each nematode was recorded and the average value was calculated. Each group was performed in triplicate. The results are shown in Table 3. Figure 2 .

[0064] Table 3. Motility of CL4176 nematode (bowing frequency, times / 30 s, mean ± SD)

[0065] In preliminary experiments on nematode lifespan, paralysis, and motor function, this invention screened seven candidate samples. The results showed that the effects of each sample on the physiological phenotype of nematodes varied significantly. Samples 1, 2, and 3 showed the most significant effects, demonstrating statistically significant differences in multiple indicators such as lifespan extension, paralysis rate reduction, and motor function improvement. Samples A, B, C, and D had relatively weak overall effects.

[0066] To further clarify the mechanism of action of key active samples and reduce redundant detection, this invention prioritizes the three most active samples for validation in subsequent molecular biology experiments, representing a group of samples with significant biological effects. This strategy improves detection efficiency while ensuring the reliability and representativeness of the mechanism study, demonstrating scientific rationality.

[0067] Test Example 4 Juglone acute oxidative stress experiment: A blank control group (Con) and treatment groups (sample 1, sample 2, and sample 3) were set up, with a drug concentration of 1.00 mg / mL for each sample. After CL4176 nematodes were synchronized to the L4 stage, they were inoculated onto NGM plates containing the corresponding samples and pretreated at 16 ℃ for 36 h, and then transferred to 25 ℃ for further culture for 24 h.

[0068] Nematodes from each group were collected, washed three times with M9 buffer, and then transferred to M9 buffer containing Juglone (final concentration 0.15 mmol / L) for incubation at 25 °C to induce acute oxidative stress. The exposure period was recorded as 0 h. The number of surviving nematodes in each group was observed and recorded every h under a stereomicroscope until all nematodes in each group died. Nematodes that did not respond to touching their heads or bodies were considered dead. At least 60 nematodes were tested in each group per experiment, and the experiment was repeated three times. Survival rates were calculated at each time point, survival curves were plotted, and the median survival time (T50) for each group was determined. The results are shown in Table 4. Figure 3 .

[0069] Table 4 Survival rate of CL4176 nematodes in each group under acute oxidative stress in Juglone (n≥60, %)

[0070] The results showed that Juglone treatment significantly reduced the survival rate of CL4176 nematodes, with almost all nematodes in the Con group dying within approximately 5 hours. In contrast, the survival rates of the Sample 1, Sample 2, and Sample 3 treatment groups all increased to varying degrees, and the survival curves shifted to the right overall. The overall protective effect was: Sample 1 > Sample 2 > Sample 3 > Con.

[0071] This indicates that the samples of the present invention can improve the tolerance of CL4176 nematodes to acute oxidative stress, with sample 1 showing the strongest antioxidant protective effect.

[0072] Experimental Example 5 To further verify the antioxidant effect of the samples of this invention at the biochemical level, the activities of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) content in the homogenate of Caenorhabditis elegans type CL4176 were determined.

[0073] Based on the aforementioned screening, Group Con and Samples 1, 2, and 3 (1.00 mg / mL) were selected as the research subjects. CL4176 nematodes were inoculated onto NGM plates with or without the samples, cultured at 16 ℃ to the L4 stage, then the temperature was increased to 25 ℃ and cultured for another 5 days to induce Aβ expression. After culture, approximately 2000 nematodes from each group were collected, washed three times with M9 buffer, the supernatant was discarded, and an appropriate amount of pre-chilled lysis buffer provided by the kit was added. The mixture was homogenized under ice bath conditions, centrifuged at 12,000 r / min for 10 min, and the supernatant was used as the sample for enzyme activity and MDA assay. Total protein content was determined using the BCA protein quantification kit.

[0074] SOD, CAT activities, and MDA content were measured using commercial kits (conventional colorimetric method) according to the manufacturer's instructions. Absorbance was measured at the corresponding wavelengths using a microplate reader, and the enzyme activity or MDA content per unit protein (U / mg prot or nmol / mg prot) was calculated. Each sample group was tested in triplicate. Results are shown in Table 5. Figure 4 .

[0075] Table 5 Effects of Acanthopanax senticosus polysaccharide and its complex on SOD, CAT activity and MDA content of CL4176 nematodes (mean ± SD, n = 3)

[0076] The results showed that, compared with the Con group, samples 1, 2, and 3 all increased the activities of SOD and CAT in the nematode homogenate to varying degrees and decreased the MDA content (Table 5). Overall, a gradient effect was observed: Sample 1 > Sample 2 > Sample 3 > Con.

[0077] Experimental results on nematode lifespan, paralysis rate, acute oxidative stress survival, SOD / CAT activity, and MDA content consistently indicate that the Acanthopanax senticosus-polysaccharide protein complex prepared in this invention can significantly improve the physiological phenotype of Caenorhabditis elegans CL4176, enhance its antioxidant defense capacity, and reduce Aβ-induced oxidative damage and neurological dysfunction.

[0078] Experimental Example 6 Given that samples 1-3 have clearly demonstrated to be the samples with the best activity in experiments 1-3, these three groups of samples were selected for subsequent molecular mechanism verification in this experiment.

[0079] Caenorhabditis elegans var. CL4176 was inoculated onto standard NGM plates and cultured at 16 °C to the L4 stage. Subsequently, the nematodes were divided into the following treatment groups: a blank control group (Con group) and treatment groups 1, 2, and 3, all administered at a concentration of 1.00 mg / mL. All treatment groups were treated on NGM plates containing the corresponding samples, maintaining the same inoculation density and culture conditions.

[0080] Each treatment group was treated at 16 °C to the L4 stage, and then transferred to 25 °C for 5 days to induce Aβ-related toxicity phenotype in CL4176 nematodes. After culture, approximately 2000 nematodes from each treatment group were collected, washed three times with M9 buffer, and transferred to EP tubes for later use.

[0081] Total RNA was extracted from nematodes using Trizol reagent according to the manufacturer's instructions. After extraction, purity (A260 / A280 and A260 / A230) was assessed using a UV spectrophotometer, and integrity was checked by 1% agarose gel electrophoresis. Once the RNA quality was confirmed, reverse transcription was performed using the BeyoRT™ First Strand cDNA Synthesis Kit to obtain the cDNA template.

[0082] Real-time quantitative PCR (qPCR) was performed using BeyoFast™ SYBR Green qPCR Mix to detect the expression level of the target gene. The qPCR reaction system and cycling conditions were performed according to the kit instructions, including pre-denaturation, cyclic amplification, and melting curve analysis. The act-1 gene was used as an internal control gene, and each sample was tested in triplicate.

[0083] The relative expression level of the target gene was calculated using the 2-ΔΔCt method, where: ΔCt = Ct(target gene) Ct (internal reference gene); ΔΔCt = ΔCt(processing group) ΔCt (control group).

[0084] By comparing the differences in ΔΔCt between each treatment group and the control group, the regulatory effects of samples 1–3 on the expression of stress-related genes (including atm-1, daf-16, jnk-1, and sod-3) in CL4176 nematodes were analyzed. The primer sequences used are shown in Table 6.

[0085] Table 6 Primer Sequences

[0086] The specific results are shown in Tables 7 to 9. Figure 5 .

[0087] Table 7. Gene expression levels of Con and Sample 1 (2-ΔΔCt, mean ± SD)

[0088] Table 8 Gene expression levels of Con and Sample 2 (2-ΔΔCt, mean ± SD)

[0089] Table 9. Con and gene expression levels in sample 3 (2-ΔΔCt, mean ± SD)

[0090] qPCR results showed that, compared with the Con group, samples 1, 2 and 3 could upregulate the expression levels of stress-related genes such as daf-16, jnk-1, sod-3 and atm-1 to varying degrees. Among them, sample 1 showed the most significant upregulation, followed by sample 2, while sample 3 had a weaker effect but still showed a certain upward trend.

[0091] In summary, the experimental results show that the Acanthopanax senticosus-polysaccharide-protein complex obtained by the microwave-enzyme synergistic extraction method of this invention has a high sugar content and good biological stability. Nematode lifespan, paralysis rate, and oxidative stress experiments showed that this substance can significantly improve the physiological phenotype of CL4176 Caenorhabditis elegans, manifested as prolonged lifespan, reduced Aβ-induced paralysis incidence, and enhanced antioxidant capacity. Juglone acute oxidative stress experiments and the results of SOD, CAT activity assays, and MDA content further confirmed that samples 1-3 can significantly enhance the antioxidant defense function of nematodes and reduce oxidative damage, consistent with the results of prolonged lifespan, reduced paralysis rate, and upregulation of genes related to stress pathways such as DAF-16 / FOXO. This invention provides experimental evidence for elucidating the biological activity mechanism of the Acanthopanax senticosus-polysaccharide-protein complex.

[0092] 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 a compound of acantho- polysaccharide protein, characterized in that, Comprising the following steps: (1) adding petroleum ether to the powder of Acanthopanax senticosus to defat, filtering and drying to obtain defatted Acanthopanax senticosus; adding citric acid-sodium citrate buffer to the defatted Acanthopanax senticosus, and then adding neutral cellulase and placing in a water bath shaker for pre-enzymolysis; (2) transferring the pre-enzymolysis system obtained in step (1) to a polytetrafluoroethylene reaction tank of a microwave extraction instrument for microwave extraction; (3) after the microwave extraction in step (2) is completed, placing the reaction tank in a boiling water bath for heating, cooling, centrifugation, and collecting supernatant I and residues; repeating the extraction once for the residues according to the above steps to obtain supernatant II; and combining the supernatants of the two times to obtain a crude extract of Acanthopanax senticosus; (4) freeze-drying the crude extract of Acanthopanax senticosus obtained in step (3) after elution with a macroporous adsorption resin and impurity removal with a dialysis bag to obtain an Acanthopanax senticosus-polysaccharide protein complex ASPS-Protein Complex.

2. The method of claim 1, wherein the preparation of the Eleutherococcus- polysaccharide protein complex is characterized by, The volume ratio of Acanthopanax senticosus to petroleum ether in step (1) is 1:5 g / mL; and the boiling range of the petroleum ether is 60-90℃.

3. The method of claim 1, wherein the preparation of the Eleutherococcus- polysaccharide protein complex is characterized by, The solid-liquid ratio of the defatted Acanthopanax senticosus to the citric acid-sodium citrate buffer in step (1) is (1:10-20) g / mL; the concentration of the citric acid-sodium citrate buffer is 0.1 mol / L, and the pH is 4.

5.

4. The method of claim 1, wherein the preparation of the Eleutherococcus- polysaccharide protein complex is characterized by, The temperature of the water bath shaker in step (1) is 40-50℃.

5. The method of claim 1, wherein the preparation of the Eleutherococcus- polysaccharide protein complex is characterized by, The addition amount of the neutral cellulase in step (2) is 1.0%, and the enzymolysis time is 30 min; the enzyme activity of the neutral cellulase is 10,000 U / g.

6. The method of claim 1, wherein the preparation of the Eleutherococcus- polysaccharide protein complex is characterized by, The microwave power in step (2) is 250-750 W, the microwave temperature is 45℃, the microwave time is 10-30 min, and the pressure is 0.1 MPa.

7. The method of claim 1, wherein the preparation of the Eleutherococcus- polysaccharide protein complex is characterized by, The heating time in the boiling water bath in step (3) is 10 min.

8. The method of claim 1, wherein the preparation of the Eleutherococcus- polysaccharide protein complex is characterized by, After the crude extract of Acanthopanax senticosus is passed through a D101 macroporous resin, dialysis is performed for 2-3 days through a 3500 Da dialysis bag, and then freeze-drying is performed to obtain an Acanthopanax senticosus-polysaccharide protein complex ASPS-Protein Complex.

9. The Acanthopanax senticosus-polysaccharide protein complex prepared by the method of any one of claims 1-8.

10. The use of the Acanthopanax senticosus-polysaccharide protein complex of claim 9 in the preparation of a drug for prolonging the lifespan of nematodes, reducing the paralysis rate of nematodes, improving the movement ability of nematodes, improving the stress resistance of nematodes, and / or inhibiting apoptosis of CL4176 Caenorhabditis elegans cells.

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