A method for extracting high-activity polysaccharide from radix codonopsis
Patent Information
- Application Number
- CN202610925569.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-25
- Publication Date
- 2026-10-27
Smart Images
Figure CN122978322A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of natural plant active ingredient extraction technology, and more specifically, to a highly efficient extraction method for Codonopsis pilosula polysaccharides. Background Technology
[0002] Codonopsis pilosula, a traditional Chinese medicine with significant medicinal and dietary value, has a long history of application in China. Its bioactive components, such as codonopsis glycosides and alkaloids, have been extensively studied. However, the fine structure and bioactivity of its polysaccharide components have not received sufficient attention. Systematically isolating and studying the structure and activity of Codonopsis pilosula polysaccharides is crucial for elucidating its medicinal value and expanding its application scope. Currently, there are various methods for extracting plant polysaccharides. While traditional hot water extraction is low-cost and simple, it relies on temperature and concentration differences to achieve polysaccharide dissolution and diffusion, resulting in a low extraction rate. Furthermore, it struggles to break down the robust network structure of plant cell walls composed of cellulose, hemicellulose, and pectin, failing to fully release polysaccharides tightly bound to cellulose. To improve extraction efficiency, modern technologies such as enzyme-assisted extraction, microwave-assisted extraction, and ultrasound-assisted extraction are gradually replacing traditional methods. Ultrasound-assisted extraction promotes mass transfer through mechanical effects, while enzyme extraction is widely used due to its mild reaction conditions and safety. Ultrasonic-assisted enzyme extraction combines the advantages of both, synergistically enhancing the effects of physical cavitation and biological enzymatic hydrolysis. Response surface methodology, as an effective mathematical and statistical method for optimizing complex processes, has been used to optimize extraction processes. However, further research is needed on the optimization of ultrasonic-assisted enzyme extraction of Codonopsis pilosula polysaccharides and on the systematic comparative study of its composition and activity with traditional hot water extraction. An efficient extraction process for Codonopsis pilosula polysaccharides needs to be established to provide a theoretical basis for its intensive processing. Summary of the Invention
[0003] To achieve the above objectives, this invention provides a method for extracting highly active Codonopsis pilosula polysaccharide (CPS), comprising raw material pretreatment, preparation of a compound enzyme preparation, ultrasound-assisted enzyme extraction (UAE), and post-treatment of the extract. (Therefore, the polysaccharide obtained by ultrasound-assisted enzyme extraction is named UAE-CPS, and the polysaccharide obtained by hot water extraction is named HWE-CPS); the ultrasound-assisted enzyme extraction and enzymatic hydrolysis processes are carried out simultaneously, and the post-treatment sequentially includes centrifugation filtration, vacuum concentration, alcohol precipitation purification, deproteinization, and freeze-drying steps.
[0004] Furthermore, the raw material pretreatment specifically involves: selecting Sichuan Codonopsis pilosula from Kaizhou, Chongqing, China, and processing it... After washing, freeze-dry, pulverize and pass through a 180-mesh sieve, and seal for storage; take the Codonopsis pilosula powder and mix it with petroleum ether at a ratio of 1:5 (w / v), reflux extract at 50℃ for 2h to remove lipids and pigments, and dry to obtain Codonopsis pilosula powder.
[0005] Furthermore, the compound enzyme preparation is made by uniformly mixing papain and cellulase at a mass ratio of 2:1, and then sealing it for later use.
[0006] Furthermore, the parameters for the ultrasound-assisted enzyme extraction are: ultrasound frequency 40kHz, ultrasound power 398.36W, ultrasound time 40.49min, liquid-to-solid ratio of the extraction system 20.49mL / g, and the amount of compound enzyme preparation added is 2.53% of the mass of Codonopsis pilosula powder.
[0007] Furthermore, the alcohol precipitation purification operation involves adding 4 times the amount of anhydrous ethanol to the concentrate, allowing it to stand at 4°C for 12 hours, and then centrifuging to separate the precipitate.
[0008] Further, the deproteinization process involves mixing polysaccharide solution, chloroform, and n-butanol in a volume ratio of 15:4:1. After thorough mixing, the bottom protein layer and organic layer are removed. This process is repeated until the protein is completely removed.
[0009] Furthermore, the freeze-drying time is 48 hours, and the extraction yield of the prepared Codonopsis pilosula polysaccharide product is 30.34%; the polysaccharide content of the Codonopsis pilosula polysaccharide is 79.67%, the protein content is 6.94%, the uronic acid content is 0.011%, and the total phenol content is 0.010%.
[0010] Furthermore, the centrifugation operation parameters after centrifugation filtration and alcohol precipitation purification are: centrifugation speed 5000 rpm, centrifugation time 5 min.
[0011] Furthermore, during the ultrasound-assisted enzyme extraction, distilled water is used as the solvent in the extraction system, and after extraction, the mixture is cooled to room temperature before centrifugation and filtration.
[0012] Furthermore, in the raw material pretreatment, Codonopsis pilosula is freeze-dried and then sealed for storage, and extracted with petroleum ether by reflux and then dried for later use, to ensure that the Codonopsis pilosula powder is free of lipid and pigment residues.
[0013] As described above, the present invention provides a method for extracting highly active Codonopsis pilosula polysaccharide (CPS), which has the following beneficial effects:
[0014] 1. This invention employs an ultrasound-assisted enzyme extraction method, using a 2:1 mixture of papain and cellulase as a composite enzyme with an enzyme addition of 2.53%. Ultrasound and enzymatic hydrolysis are carried out simultaneously. The ultrasonic cavitation effect physically breaks down the cell wall, while the enzyme specifically degrades structural components. The two work synergistically to achieve a triple effect of physical disruption, biochemical degradation, and enhanced mass transfer, increasing the extraction rate to 30.34%, which is significantly higher than traditional methods and reduces resource waste.
[0015] 2. This invention avoids the excessive dissolution of impurities, resulting in a finished product with a polysaccharide content of 79.67%, a protein content reduced to 6.94%, and uronic acid and total phenol contents of only 0.011% and 0.010%, respectively, effectively improving product purity.
[0016] 3. The ultrasound-assisted enzyme extraction temperature of this invention is controlled at 40~60℃, the extraction time is short, and the process parameters are optimized by response surface methodology to avoid over-processing. FT-IR and NMR analyses confirm that the polysaccharide functional groups are not destroyed and still maintain the pyranose-type structure and α-glycosidic bonds. In vitro antioxidant experiments show that its IC50 values for ABTS free radicals and hydroxyl free radicals are 1.79 mg / mL and 0.81 mg / mL, respectively, which are significantly better than those of products produced by traditional methods, and have stronger antioxidant activity.
[0017] 4. The ultrasonic cavitation effect and shear force of this invention destroy the polysaccharide aggregation structure, and the enzyme degradation of protein impurities avoids polysaccharide cross-linking. Combined with the subsequent freeze-drying process, the solubility and dispersibility are significantly improved, and the higher zeta potential indicates better antibacterial properties, thus expanding the application scenarios in the food and pharmaceutical fields.
[0018] 5. The process of this invention has strong stability and good repeatability, the operation process is clear, the required instruments are easy to obtain, and it is suitable for industrial-scale production. Attached Figure Description
[0019] Figure 1 This is the standard curve for each component.
[0020] Figure 2 It is a single-factor optimization of UAE-CPS.
[0021] Figure 3 It is a three-dimensional and contour plot of the interactions between different variables in the UAE-CPS model.
[0022] Figure 4 These are the FT-IR spectrum and NMR 13C spectrum of CPS.
[0023] Figure 5 These are scanning electron microscope images of HWE-CPS and UAE-CPS.
[0024] Figure 6 It is the in vitro antioxidant activity of CPS. Detailed Implementation
[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments:
[0026] like Figures 1 to 6As shown, a method for extracting highly active Codonopsis pilosula polysaccharide (CPS) includes raw material pretreatment, preparation of a compound enzyme preparation, ultrasound-assisted enzyme extraction, and post-treatment of the extract. The ultrasound-assisted enzyme extraction and enzymatic hydrolysis processes are carried out simultaneously. The post-treatment sequentially includes centrifugation and filtration, vacuum concentration, alcohol precipitation purification, deproteinization, and freeze-drying. Specifically, the raw material pretreatment involves selecting Codonopsis pilosula from Kaizhou, Chongqing, China, and then... After washing, the powder was freeze-dried, pulverized, and passed through a 180-mesh sieve, then sealed and stored. The Codonopsis pilosula powder was mixed with petroleum ether at a ratio of 1:5 (w / v), and refluxed at 50°C for 2 hours to remove lipids and pigments. After drying, the Codonopsis pilosula powder was obtained. The compound enzyme preparation was prepared by uniformly mixing papain and cellulase at a mass ratio of 2:1, and then sealed for later use. The parameters for the ultrasound-assisted enzyme extraction were: ultrasound frequency 40kHz, ultrasound power 398.36W, ultrasound time 40.49min, liquid-to-solid ratio of 20.49mL / g, and the amount of compound enzyme preparation added was 2.53% of the mass of the Codonopsis pilosula powder. The alcohol precipitation purification operation involved adding 4 times the amount of anhydrous ethanol to the concentrated solution, allowing it to stand at 4°C for 12 hours, and then centrifuging to separate the precipitate. The deproteinization treatment involved mixing polysaccharide solution, chloroform, and n-butanol at a volume ratio of 15:4:1, thoroughly mixing, and then removing the bottom protein layer and organic layer. This process was repeated until the protein was completely removed. The freeze-drying time was 48 hours, and the yield of the prepared Codonopsis pilosula polysaccharide product (UAE-CPS) was 30.34%. The polysaccharide content of the Codonopsis pilosula polysaccharide was 79.67%, the protein content was 6.94%, the uronic acid content was 0.011%, and the total phenol content was 0.010%. The centrifugation parameters after centrifugation filtration and alcohol precipitation purification were: centrifugation speed 5000 rpm, centrifugation time 5 min. During the ultrasound-assisted enzyme extraction, distilled water was used as the solvent. After extraction, the mixture was cooled to room temperature before centrifugation filtration. After freeze-drying, Codonopsis pilosula was sealed and stored. After reflux extraction with petroleum ether, it was dried for later use to ensure that the Codonopsis pilosula powder was free of lipid and pigment residues.
[0027] 1. Codonopsis pilosula is a traditional Chinese medicine and food therapy product containing active ingredients such as codonopsis glycosides. The fine structure and bioactivity of its polysaccharides require further investigation. Composed of monosaccharides such as glucose, some studies have confirmed its antioxidant activity. Various methods exist for extracting plant polysaccharides. Traditional hot water extraction has low extraction rates, while modern technologies such as enzyme-assisted and ultrasound-assisted extraction are more efficient. The combination of ultrasound and enzymes can synergistically disrupt plant cell walls, improving extraction efficiency. This study uses a Box-Behnken response surface methodology to optimize the process parameters for ultrasound-assisted enzyme extraction of Codonopsis pilosula polysaccharides. Comparison with traditional hot water extraction methods was conducted to analyze the differences in product composition, antioxidant activity, and structure, providing theoretical support for the intensive processing of Codonopsis pilosula.
[0028] 2. Materials and Methods
[0029] 2.1: Medicines and Instruments
[0030] The Sichuan Codonopsis pilosula, sourced from Kaizhou, Chongqing, China, was washed with H2O, freeze-dried, pulverized, sieved through a 180-mesh sieve, sealed, and stored. The powder was then mixed with petroleum ether at a ratio of 1:5 (w / v) and refluxed at 50°C for 2 hours to remove lipids, pigments, etc., before drying for later use.
[0031] Experimental instruments: IR408 Fourier transform infrared spectrometer (Shimadzu, Japan), UV-2550 ultraviolet-visible spectrophotometer (Shimadzu Instruments), HXLG-12-80D vacuum freeze dryer (Shanghai Huxi Experimental Co., Ltd.), TG-20 high-speed centrifuge (Sichuan Shuke Instrument Co., Ltd.).
[0032] 2.2: HWE of CPS
[0033] 10g of Codonopsis pilosula powder was placed in an Erlenmeyer flask and extracted with hot water. After extraction, the extract was cooled, centrifuged, and filtered to obtain a polysaccharide extract. The extract was then distilled under reduced pressure to a suitable volume. Four times the volume of anhydrous ethanol was added, and the solution was placed in a refrigerator set at 4°C for approximately 12 hours. The solution was centrifuged at 5000 rpm for 5 minutes to separate the extract. Approximately 10 mL of H₂O was added to dissolve the extract, which was then frozen into a solid and freeze-dried for 48 hours to obtain HWE-CPS. The yield was calculated. The yield of CPS was calculated using the following formula:
[0034] (2.1)
[0035] 2.3: UAE of CPS
[0036] 10g of Codonopsis pilosula powder was mixed with a certain amount of H2O in a beaker and extracted with ultrasound at a frequency of 40kHz. After the ultrasound was completed, an appropriate amount of enzyme was added for enzymatic hydrolysis. After the enzymatic hydrolysis was completed, the mixture was cooled to room temperature, centrifuged and filtered to obtain polysaccharide extract. The subsequent steps were carried out according to the method in 2.2 to obtain UAE-CPS. The yield of UAE-CPS was calculated by formula (2.1).
[0037] 2.3. UAE Optimization of CPS
[0038] 2.3.1: UAE Single-Factor Experiment
[0039] This experiment will investigate the relationship between CPS yield and temperature, ultrasonic power, and ultrasonic time. 10g of Codonopsis pilosula powder was weighed and placed in a beaker. UAE-CPS was extracted according to the experimental method in section 2.3, and its yield was calculated. The optimal ultrasonic time was explored under experimental conditions of 10, 20, 30, 40, 50, and 60 min, while keeping the temperature, liquid-to-solid ratio, ultrasonic power, and enzyme level constant. The optimal values for single-factor experiments were investigated by keeping the ultrasonic time, ultrasonic power, and enzyme level constant; experiments were conducted at 40, 45, 50, 55, 60, and 65℃. Under the condition of keeping the temperature, enzyme level, and ultrasonic time constant constant, the optimal values of ultrasonic power in the single-factor experiments were investigated, which were 300, 350, 400, 450, 500, 550, 600, and 650 W, respectively. Under the condition of keeping the temperature, enzyme level, and ultrasonic time constant, the optimal value of enzyme ratio in the single-factor experiments was investigated (the optimal extraction rate of cellulase + papain had been previously determined experimentally). The single-factor experimental design is shown in Table 1.
[0040]
[0041] 2.3.2: RSM Experiment of UAE-CPS
[0042] Based on the Box-Behnken principle, the four factors of required liquid-to-material ratio, ultrasonic time, ultrasonic power, and enzyme ratio were organized as variable conditions, and the experimental design shown in Table 2 was carried out to mainly investigate the comprehensive influence of these four factors on the yield.
[0043]
[0044] 2.4: CPS deproteinization
[0045] HWE-CPS and UAE-CPS are mixtures containing proteins. Therefore, deproteinization is necessary. Among polysaccharide deproteinization methods, the Sevag method is relatively common. A CPS solution, chloroform, and n-butanol in a volume ratio of 15:4:1 are added to a separating funnel and mixed thoroughly for approximately 15 minutes. The protein and organic layers at the bottom are removed, yielding the supernatant. The supernatant is then subjected to alcohol precipitation and resolubilization according to the experimental method described in section 2.2. After removing the proteins, the CPS is freeze-dried.
[0046] 2.5: Identification of CPS Chemical Composition
[0047] The CPS composition obtained by the optimized process described in 2.4 and 2.5 contains not only polysaccharides but also proteins and other substances, requiring analysis of its components. The concentrations of the standard solutions to be tested were set to 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL, where "y" represents the absorbance at different wavelengths and "x" represents the concentration of the component.
[0048] 2.5.1: Identification of Polysaccharide Content
[0049] This experiment used the phenol sulfate method to calculate the polysaccharide content in CPS. For example... Figure 1 As shown in (a), the linear regression equation for the glucose standard curve is:
[0050] y = 5.4853x + 0.2445 (2.2)
[0051] 2.5.2: Identification of Protein Content
[0052] This experiment used the Commus Brilliant Blue staining method to determine the CPS protein content; such as Figure 1 As shown in (b), the figure contains the linear regression equation for the bovine serum albumin standard curve:
[0053] y = 0.5859x + 0.176 (2.3)
[0054] 2.5.3: Determination of uronic acid content
[0055] This experiment used the sulfuric acid-carbazole method to determine the uronic acid content in CPS, such as... Figure 1 As shown in (c), the linear regression equation for the uronic acid standard curve is:
[0056] y = 4564x + 0.5956 (2.4)
[0057] 2.5.4: Determination of total phenol content
[0058] This experiment is based on the determination of total phenol content in CPS using the forintol method, such as Figure 1 As shown in (d), the linear regression equation for the gallic acid standard curve is:
[0059] y = 0.703x + 1.6248(2.5)
[0060] 2.6: Structural Analysis
[0061] 2.6.1: FT-IR Spectroscopic Analysis
[0062] Approximately 4 mg each of HWE-CPS and UAE-CPS were taken, and FT-IR analysis was performed on the two polysaccharides separately. The scan range was set to... .
[0063] 2.6.2: NMR Analysis
[0064] Approximately 50 mg of HWE-CPS and UAE-CPS were dissolved in 0.45 mL of heavy water, centrifuged, and the supernatant was collected into an NMR tube. The NMR spectrometer was 300 MHz C13CPD.
[0065] 2.7. Morphological Analysis
[0066] Weigh approximately 5-10 mg of HWE-CPS and UAE-CPS powders separately and dissolve them in 10 mL of anhydrous ethanol. Place the solution in an ultrasonic cleaner and ultrasonically disperse for 15-20 minutes to ensure the polysaccharides are fully and uniformly dispersed in the solvent, forming a homogeneous suspension. Use tweezers to take a small piece of conductive double-sided tape and smoothly attach it to the center of the sample holder. Gently press it with your hand or tweezers to ensure good contact with the sample holder and no air bubbles. Use a pipette to carefully and slowly drop 2-5 μL of the thoroughly mixed polysaccharide dispersion onto the conductive tape. Place the sample holder in a clean petri dish, cover it with filter paper, and allow it to air dry at room temperature for at least 2-4 hours to allow the ethanol to completely evaporate. After uniformly spraying a gold film with a thickness of approximately 10-20 nm onto the sample surface, insert it into the sample chamber of a scanning electron microscope for image acquisition.
[0067] 2.8. Zeta potential and particle size analysis
[0068] Weigh 2 mg of polysaccharide and dissolve it in 10 mL of distilled water to prepare a 0.1 mg / mL solution. Measure its potential on a dynamic light scattering instrument and analyze its zeta potential and particle size.
[0069] 2.9. Solubility determination
[0070] Weigh 5g each of HWE-CPS and UAE-CPS and dissolve them in 10mL of distilled water to prepare saturated solutions containing undissolved solute. Dissolve the solutions by magnetic stirring at 20℃ (room temperature) and 60℃ for 2 hours respectively. Centrifuge, discard the supernatant, dry the remaining polysaccharides to constant weight, weigh the remaining solute, and calculate their solubility using the following formula:
[0071] (2.6)
[0072] In the formula: The mass of the polysaccharide weighed before dissolution;
[0073] The mass of the polysaccharide dried to constant weight;
[0074] The volume of the solvent;
[0075] 2.10: In vitro antioxidant activity analysis of CPS
[0076] The absorbance values of HWE-CPS and UAE-CPS reaction solutions at specific wavelengths were determined by ultraviolet spectrophotometry to assess their antioxidant capacity from four aspects. Ascorbic acid (Vc) solution was used as a positive control in all subsequent antioxidant experiments.
[0077] 2.10.1: Determination of CPS's ability to scavenge DPPH free radicals
[0078] The DPPH free radical scavenging capacity was determined according to the literature. Based on the actual situation, 2 mL of CPS solution of different concentrations was added to the same volume of DPPH-ethanol solution (0.5 mmol / L). The reaction was carried out in the dark for approximately 30 min, and the absorbance value was measured at 517 nm. The DPPH free radical scavenging capacity of different concentrations of HWE-CPS and UAE-CPS was calculated as follows:
[0079] (2.4)
[0080] In the formula: This represents the absorbance value of the sample solution.
[0081] The absorbance value is for anhydrous ethanol instead of DPPH;
[0082] The absorbance value is given by the solvent instead of the sample solution.
[0083] 2.10.2: Determination of CPS's ability to scavenge hydroxyl radicals
[0084] Take 1 mL of CPS solutions of different concentrations, add equal amounts of FeSO4 (10 mmol / L), salicylic acid-ethanol solution (10 mmol / L), and H2O2 (10 mmol / L), mix well, and react in a 40°C water bath for 25 min. Measure the absorbance at 510 nm. The hydroxyl radical scavenging ability of different concentrations of CPS is calculated as follows:
[0085] (2.5)
[0086] In the formula: This represents the absorbance value of the sample solution.
[0087] The absorbance value is obtained by replacing the colorimetric mixture with anhydrous ethanol.
[0088] The absorbance value is given by the solvent instead of the sample solution.
[0089] 2.10.3: Characterization of CPS's ability to scavenge ABTS free radicals
[0090] Following the same method with slight modifications, an ABTS diammonium salt solution (7.4 mmol / L) and a K₂S₂O₈ solution (2.6 mmol / L) were prepared. Equal volumes of these solutions were mixed and added to a brown bottle, then stored in the dark for 12 hours. The absorbance was adjusted to 0.7 at 734 nm using phosphate buffer (pH = 7.2-7.4) to obtain the ABTS radical scavenging solution. 1 mL of different concentrations of CPS were mixed with 3 mL of the ABTS radical scavenging solution and reacted in the dark for 10 minutes. The absorbance was then measured at 734 nm. The ABTS radical scavenging capacity of different concentrations of CPS was calculated as follows:
[0091] (2.6)
[0092] In the formula: This represents the absorbance value of the sample solution. The absorbance value is obtained by replacing the colorimetric mixture with anhydrous ethanol. The absorbance value is given by the solvent instead of the sample solution.
[0093] 2.11: Analysis of the in vitro antibacterial activity of CPS
[0094] Polysaccharide solutions with concentrations of 0, 10, 20, 30, and 40 mg / mL were prepared. The polysaccharide solutions of different concentrations were co-incubated with Escherichia coli and Staphylococcus aureus with adjusted bacterial concentrations for 10 min. The co-incubated bacterial solutions were evenly spread on agar medium and incubated for 8 h at 25°C and humidity greater than 60%. The number of colonies was then counted.
[0095] 3. Results and Discussion
[0096] 3.1: Single-factor optimization
[0097] 3.1.1: Single-factor optimization of UAE-CPS
[0098] Under a fixed liquid-to-solid ratio of 20 mL / g, the optimal conditions for six single factors—temperature, ultrasonic power, ultrasonic time, enzyme type, enzyme level, and enzyme ratio—were explored. One factor was changed while the other five remained constant, and the yield of HWE-CPS was compared. The horizontal axis represents the single factor settings at different gradients, and the vertical axis represents the yield of UAE-CPS; for example... Figure 2 As shown. From Figure 2 (a) It can be seen that the yield begins to decrease when the temperature exceeds 55°C. It is generally believed that excessively high temperatures will inhibit enzyme activity or even deactivate the enzyme, and will promote polysaccharide hydrolysis. When the temperature exceeds 55°C, the enzyme is deactivated and the polysaccharide is hydrolyzed, thereby reducing the yield. Figure 2(b) indicates that when the ultrasonic time is less than 40 min, the yield is significantly increased. This may be because the longer cavitation time disrupts the cell walls of the raw material, thereby reducing the binding of polysaccharides to structures such as cell walls and promoting polysaccharide release. Beyond 40 min, the yield suddenly begins to decline. Assuming that the solubility of polysaccharides has reached its maximum at this point, excessively long ultrasonic times have no effect on improving the yield and may even lead to polysaccharide hydrolysis. After exceeding the optimal time of 40 min, the releaseable polysaccharides are nearly complete, and the release rate tends to zero, while the degradation process continues. This results in the amount of measurable macromolecular polysaccharides in the solution no longer increasing or even decreasing, leading to a plateau or decline in the extraction rate. Figure 2 (c) It can be seen that increasing ultrasonic power has an impact on improving the yield of UAE-CPS within a certain range. Higher power increases cavitation and system pressure, thereby increasing the possibility of polysaccharides breaking down cell walls and releasing solubilizing agents. However, the cavitation effect generated by excessive ultrasonic power produces reactive oxygen species such as hydroxyl radicals. The higher the power, the greater the cavitation intensity, and the more exponentially the free radicals are generated. These high-concentration free radicals will aggressively attack polysaccharide chains, oxidize sugar rings, break glycosidic bonds, and irreversibly degrade large polysaccharides into oligosaccharides or even monosaccharides, thereby reducing the yield. Figure 2 (d) It is known that the combination of cellulase and papain yields the highest polysaccharide recovery. Enzyme-assisted extraction increases polysaccharide yield by dissolving cell walls, causing cell sap to flow out. Some plant cell walls contain abundant hydroxyproline glycoproteins, which covalently cross-link with polysaccharides (such as pectin and cellulose), forming an extremely robust protein-polysaccharide network. This network is like reinforced concrete, much stronger than a simple pectin-cellulose wall. Papain specifically hydrolyzes the peptide bonds of these structural proteins. Once the protein cross-links are broken, the entire cell wall structure instantly disintegrates, exposing cellulose and pectin. This indicates that the cell walls of *Codonopsis pilosula* contain abundant hydroxyproline glycoproteins, allowing papain to more effectively break down the cell walls and promote polysaccharide extraction. Figure 2 (e) It is known that the polysaccharide extraction rate is highest at an enzyme level of 2.0%. Enzyme-driven polysaccharide extraction is achieved by specifically destroying the cell wall, forcing the polysaccharide to flow out of the cell. The cell wall components (cellulose, structural proteins, etc.) in the raw material that can be contacted and hydrolyzed by the enzyme are limited. Once the enzyme quantity is sufficient to complete the hydrolysis of these accessible substrates within a set time, no amount of enzyme can hydrolyze non-existent substrates. Figure 2(f) It can be seen that the polysaccharide extraction rate is the highest when the ratio of cellulase to papain is 1:2. In the optimal ratio, the amount of papain is just enough to efficiently hydrolyze the structural proteins in the cell wall as "cross-linking agents" in the early stage of the reaction. The appropriate amount of cellulase can make full use of the "window period" created by the protease and the "fragmented" cellulose substrate, thereby achieving the purpose of breaking the cell wall. This also explains the main components and ratio of the cell wall of Codonopsis pilosula.
[0099] 3.2: RSM Optimization and Results
[0100] 3.2.1: RSM optimization and results of UAE-CPS
[0101] Based on the results of the single-factor experiments, a four-factor, three-level experimental design was conducted using the Box-Behnken central composite design. This design selected four factors as variables: liquid-to-solid ratio (A), sonication time (B), sonication time (C), and enzyme level (D), with R representing the yield of UAE-CPS. A total of 29 exploratory experiments were conducted. The experimental design is shown in Table 3.
[0102]
[0103]
[0104] Table 3. RSM experimental design for CPS
[0105] (1) Fitting and analysis of variance of UAE-CPS regression equation
[0106] By fitting the data in Table 8, a mathematical model is established, and the quadratic regression equation is determined: (3.1)
[0107] Where x represents the liquid-to-material ratio; y represents the ultrasonic power; z represents the ultrasonic time; and m represents the enzyme level.
[0108] Table 4 shows that, based on the principle that a larger f-value has a greater impact on the yield of UAE-CPS, the order of influence of A: liquid-to-solid ratio, B: ultrasonic power, C: ultrasonic time, and D: enzyme level on the yield of UAE-CPS is D > A > C > B. The pure error of this model is not significant, with an f-value of 256.61 and a p-value < 0.0001, indicating a very good significance effect. This suggests that the experimental results predicted by this model have a high probability of matching the actual results. Finally, the model constructions that have a significant impact on the extraction rate include A, C, D, AC, AD, BC, BD, CD, ... , , , The coefficients of the quadratic terms in this equation are mostly negative, indicating that the response surface curve opens downwards and has a maximum response value. Table 5 shows that... =0.9961, adjust =0.9922, with a difference of 0.0039, indicating that the experimental model results fit well and the data are reliable. This can be used as the optimal extraction condition for analyzing UAE-CPS and predicting its optimal extraction rate.
[0109]
[0110]
[0111] Table 4. Analysis of Variance for CPS Regression Model
[0112]
[0113] Table 5. Analysis of Variance Regression Model for CPS
[0114] (2) Interaction between UAE-CPS factors
[0115] Combination Figure 3 The interaction and influence between two factors were analyzed. The steeper the 3D curve, the more elliptical the profile shape, and the denser the profile, the more significant the interaction between the two factors and the greater the impact on yield. Figure 5 Steeper 3D curves in (b, c, g, h) and Figure 3 The near-elliptical shape of the contour lines in (e, f, i, j) indicates that A: the interaction between liquid-to-solid ratio and C: ultrasonic time is significant; A: the interaction between liquid-to-solid ratio and D: enzyme level is significant; B: the interaction between ultrasonic power and C: ultrasonic time is significant; and B: the interaction between ultrasonic power and D: enzyme level is significant. The p-values < 0.0001 in Table 4 also support this finding. Meanwhile, from... Figure 3 As can be seen from (a-1~2) and (g1~2), the interaction between A: liquid-to-solid ratio and B: ultrasonic power, and between C: ultrasonic time and D: enzyme level is not significant. The p value in Table 5 > 0.05 is consistent with this result.
[0116] (3) Determination and verification of optimal process conditions for UAE-CPS
[0117] Based on the response surface methodology analysis, the optimal process for UAE-CPS was predicted to be a liquid-to-solid ratio of 20.49 mL / g, an ultrasonic time of 40.49 min, an ultrasonic power of 398.36 W, an enzyme level of 2.53%, and a yield of 30.34%. With the premise of ease of operation, the predicted optimal process conditions were appropriately adjusted. Three parallel experiments were conducted under the conditions of ultrasonic power of 398 W, a liquid-to-solid ratio of 20 mL / g, an ultrasonic time of 40 min, and an enzyme level of 2.5%, with an average yield of 30.12%. The relative error between this yield and the theoretical optimal yield was 0.72%, demonstrating the reliability of the experimental model.
[0118] 3.2.2: Comparison of HWE-CPS and UAE-CPS
[0119] To determine the difference in the effects of hot water extraction (HWE) and ultrasound-assisted enzymatic extraction (UAE) on the extraction rate of Codonopsis pilosula polysaccharides (CPS), an independent samples t-test was used to statistically analyze the parallel experimental data of the two processes. A highly significant difference was found in the extraction rates between the UAE and HWE methods (P < 0.01), with the UAE method (30.12%) showing a significantly higher extraction rate than the HWE method (20.07%). This demonstrates that UAE can effectively improve the extraction rate of Codonopsis pilosula polysaccharides, thereby reducing waste. This result is because traditional hydrothermal extraction mainly relies on high temperatures to promote polysaccharide dissolution and diffusion, which has limited impact on the dense structure of plant cell walls. In contrast, ultrasound propagation in liquids generates countless tiny bubbles (cavitation bubbles). These bubbles violently collapse under sound pressure, instantly generating extremely high local temperatures and strong shock waves / microjets. This extreme physical force can directly tear and break plant cell walls, creating micropores and cracks, and even breaking large particles into smaller ones, greatly increasing the contact area between the solvent and intracellular polysaccharides, thus exposing deeply bound polysaccharides. Simultaneously, the added cellulase and pectinase precisely catalyze the hydrolysis of structural components of the cell wall and membrane, releasing deep polysaccharides encapsulated by physical barriers, resulting in more thorough extraction. Furthermore, the enzymes exhibit high regional selectivity and specificity in their degradation, gently opening the cell wall network without indiscriminately damaging the target active structure of the polysaccharides. Ultrasonic-assisted enzyme extraction combines the advantages of both methods. Ultrasound not only disrupts the structure itself but also promotes contact between the enzyme and the substrate (cell wall), accelerating the enzyme reaction rate. It may also alter the conformation of enzyme molecules, making their active sites more readily exposed and increasing enzyme activity. The mechanical effect and perturbation of ultrasound strongly stir the solution system, eliminating concentration gradients at the extraction interface and greatly enhancing the mass transfer process. This allows dissolved polysaccharides to rapidly diffuse into the bulk solvent while fresh solvent continuously impacts the raw material surface. This enhanced mass transfer allows the extraction process to reach equilibrium faster, and the equilibrium point (i.e., the final extraction rate) is higher. In summary, the high extraction rate of ultrasound-assisted enzyme extraction is not due to a single factor, but rather the result of the synergistic effect of "physical disruption + biochemical degradation + enhanced mass transfer".
[0120] 3.3: Component Analysis of HWE-CPS and UAE-CPS
[0121] Prepare 0.5 mg / mL HWE-CPS and UAE-CPS solutions, and calculate the absorbance of the reaction solutions at each wavelength according to the experimental method in section 2.7. (Refer to...) Figure 1The regression curves were used to calculate the content of each component in HWE-CPS and UAE-CPS extracted under optimal extraction conditions. As shown in Table 12, polysaccharides were the dominant component in CPS extracted by both methods, followed by proteins, while phenolic substances were the least abundant. The comparison in Table 12 shows that the polysaccharide content of UAE-CPS was higher than that of HWE-CPS, with the former at 78.89% and the latter at 69.24%. The slightly higher sugar content in UAE-CPS indicates that the ultrasound-assisted enzyme extraction method is slightly superior to the hot water extraction method in terms of polysaccharide content.
[0122] 3.3.1: Comparison of HWE-CPS and UAE-CPS components
[0123] To determine the differences in the effects of hot water extraction (HWE-CPS) and ultrasound-assisted enzyme extraction (UAE-CPS) on the sugar and protein content of CPS polysaccharides, an independent samples t-test was used to statistically analyze the parallel experimental data of the two extraction methods. A highly significant difference was found in the sugar content of polysaccharides extracted by the UAE method and the HWE method (P < 0.01), with the sugar content of polysaccharides extracted by UAE-CPS being significantly higher than that extracted by HWE-CPS. However, a highly significant difference was found in the protein content of polysaccharides extracted by UAE-CPS and HWE-CPS (P < 0.01), with the protein content of polysaccharides extracted by UAE-CPS being significantly lower than that extracted by HWE-CPS. These results are attributed to the synergistic effect of ultrasound and enzymes: the cavitation effect of ultrasound increases the contact area between the raw material and the enzyme, improving the catalytic efficiency of the enzyme; simultaneously, the gentle thermal effect generated by ultrasound maintains enzyme activity (avoiding enzyme inactivation due to high temperatures), further enhancing the extraction effect. Hot water extraction typically requires higher temperatures and longer extraction times. High temperatures can lead to the degradation of some polysaccharides, resulting in polysaccharide loss. Furthermore, the longer extraction time causes the leaching of proteins and pectins, increasing impurities in the polysaccharides and further reducing the final sugar content, while potentially increasing the protein content. These results align with the initial objectives of this study; UAE not only significantly improves the extraction rate but also yields products with higher levels of active ingredients compared to traditional extraction methods.
[0124]
[0125] Table 6. Component analysis of HWE-CPS and UAE-CPS
[0126] 3.4. Structural Analysis
[0127] 3.4.1.FT-IR analysis
[0128] Infrared spectroscopy is a commonly used method for detecting the functional groups of polysaccharides. The absorption peaks of the characteristic functional groups of polysaccharides are mostly located in... The strong absorption peaks of HWE-CPS and UAE-CPS are located at [locations missing]. and This is caused by the stretching vibration of the non-free hydroxyl groups in the polysaccharide chain, and the two polysaccharides are in The two polysaccharides exhibit identical absorption peaks, which are generated by the stretching vibrations of the CH bonds of the methyl and methylene groups in the polysaccharides. No C=O absorption peak was found in the 1700–1750 cm⁻¹ range, suggesting that both polysaccharides may contain small amounts of uronic acid, or they may not contain uronic acid at all. This hypothesis is consistent with the results obtained in Table 12. place and The absorption peak at 1426.9 and 1417.6 is generated by the water bound to the polysaccharide. The weak peaks at 1426.9 and 1417.6 may be due to the angular vibration of CH. Pyranoses often have continuous absorption peaks in the range of 1200-1000. HWE-CPS in There is a weak absorption peak on it. There is a strong absorption peak on it, UAE-CPS in There is a faint absorption peak on it. Strong absorption peaks were observed on the surface. This indicates that both samples contain COH and COC functional groups. The two polysaccharides exhibited... place and An absorption peak appeared at the same location, which is a characteristic peak of the furanose ring, but it was also present at... The absence of a split-shaped medium-intensity peak nearby suggests that the CPS may not contain furanose rings or may contain only a small amount of furanose rings, which is consistent with the NMR spectrum. and The weak absorption peaks at [specific locations] indicate the possible presence of β-glycosidic bonds in both polysaccharides, which was confirmed by the NMR analysis in section 3.5.2. Both HWE-CPS and UAE-CPS are pyranoses. The functional groups of HWE-CPS and UAE-CPS are essentially the same, indicating that ultrasonic extraction of polysaccharides under the parameters determined in this study does not damage the polysaccharide structure and can effectively improve the extraction rate.
[0129] 3.4.2: NMR Analysis
[0130] Figure (b) shows the carbon NMR spectrum of CPS. The signal peaks at 103.66 ppm and 103.11 ppm are generally associated with carbon atoms (C1 atoms) in the α- or β-glycosidic bonds of the sugar, indicating that the polysaccharide contains monosaccharide units. The signal peaks of C2-C5 on the sugar ring are mainly concentrated in the region of 69.30-81.08 ppm. The heterohead carbon signal peaks show multiple C1 peaks with gradually decreasing signals (103.66 ppm, 103.11 ppm, 98.13 ppm, 92.46 ppm), indicating that CPS may contain multiple different C1 peak monosaccharide residues. The number of monosaccharide residues producing resonance peaks at 98.13 ppm and 92.46 ppm is relatively low compared to the number of monosaccharide residues producing a resonance peak at 103.66 ppm. Also, based on the fact that the chemical shift of 90-102 ppm is an α-glycosidic bond, it can be inferred that Codonopsis pilosula polysaccharide mainly contains α-glycosidic bonds. Between 60 and 110 ppm, except for the signal from heterotopic carbon atoms, all other signal peaks were less than 80 ppm, indicating that Codonopsis pilosula polysaccharides consist of pyran rings with little or no furan ring involvement, which is consistent with the FT-IR results. Additionally, the signal peak at 60 ppm is presumably generated by its C6 carbon. Furthermore, no signal peaks were found in the 160–180 ppm range, indicating that NMR did not detect the carboxyl carbon signal of uronic acid, consistent with the low uronic acid content in CPS.
[0131] 3.5: Morphological Analysis
[0132] Figure a shows a scanning electron microscope (SEM) image of polysaccharides obtained by hydrothermal extraction. It can be seen that the polysaccharides mostly exhibit large, blocky, or thick, lamellar aggregates with relatively smooth surfaces and dense structures. This is mainly because under the high temperature and pressure extraction environment, the movement of polysaccharide molecular chains intensifies, and intermolecular forces such as hydrogen bonds facilitate rearrangement and tight cross-linking, leading to severe aggregation. This dense structure may affect its solubility and bioavailability. In contrast, the polysaccharides in Figure b (ultrasound-assisted enzymatic extraction) exhibit a more ideal porous, loose fibrous network structure. This morphology indicates that the cavitation effect of ultrasound and the specific degradation by enzymes work synergistically, effectively disrupting plant cell walls, allowing for more complete release of polysaccharides, and creating a large specific surface area. This loose, porous structure usually indicates better solubility, adsorption, and potential biological activity.
[0133] 3.6 ζ-potential and particle size analysis
[0134] The average values of the zeta potential and particle size of HWE-CPS and UAE-CPS from parallel experiments are shown in Table 7.
[0135] 3.6.1 Comparison of zeta potentials between HWE-CPS and UAE-CPS
[0136] To clarify the difference in the effects of HWE and UAE on the zeta potential of CPS, an independent samples t-test was used to statistically analyze the parallel experimental data of the two extraction methods. The results showed a highly significant difference in the zeta potential between HWE-CPS and UAE-CPS (P < 0.01), with the zeta potential of UAE-CPS being significantly higher than that of HWE-CPS. This result is attributed to the cavitation effect of ultrasound, which leads to more uniform dispersion of polysaccharide molecules and reduces charge cancellation caused by aggregation. Furthermore, the enzyme can degrade electrically neutral impurities such as proteins and pectin bound to polysaccharides, reducing the "shielding" of polysaccharide surface charges by these impurities and allowing for more complete exposure of the polysaccharide surface charges. This results in a significantly higher zeta potential for UAE-CPS compared to HWE-CPS, suggesting that the antibacterial activity of UAE-CPS is superior to that of HWE-CPS.
[0137] 3.6.2: Comparison of particle size between HWE-CPS and UAE-CPS.
[0138] To clarify the differences in the effects of HWE and UAE on the particle size of CPS, an independent samples t-test was used to statistically analyze the parallel experimental data of the two extraction methods. The results showed a highly significant difference in polysaccharide particle size between HWE-CPS and UAE-CPS (P < 0.01), with the particle size of HWE-CPS being significantly larger than that of UAE-CPS. This is because the cavitation effect of ultrasound can directly impact polysaccharide molecular aggregates, breaking them into smaller particles; simultaneously, the shear force of ultrasound can sever non-covalent bonds such as hydrogen bonds and hydrophobic interactions between polysaccharide molecules, preventing polysaccharide chains from entangled and forming large aggregates. Furthermore, tebuconazole can degrade protein impurities bound to polysaccharides, reducing the possibility of proteins acting as "bridges" connecting polysaccharide molecules to form large particles. Hot water extraction, relying solely on high temperatures to dissolve polysaccharides, cannot effectively break down the aggregate structure between polysaccharide molecules, resulting in extracted polysaccharides existing in the form of larger aggregates. Smaller particle size usually indicates better solubility.
[0139]
[0140] Table 7 Zeta potential and particle size of CPS
[0141] 3.7. Solubility
[0142] Table 8 shows the average solubility of HWE-CPS and UAE-CPS at room temperature and 60℃. Both HWE-CPS and UAE-CPS are readily soluble in water. To clarify the difference in the effects of HWE and UAE on the particle size of CPS, an independent samples t-test was used to statistically analyze the parallel experimental data of the two extraction methods. The results showed a highly significant difference in room temperature solubility between UAE-CPS and HWE-CPS (P < 0.01), and the room temperature solubility of UAE-CPS was significantly higher than that of HWE-CPS. This result is consistent with the results of SEM and particle size prediction. The loose and porous microstructure and large specific surface area allow UAE-CPS to contact water better, and the smaller particle size indicates that the intermolecular forces of UAE-CPS are weaker, making it easier to dissolve in water.
[0143]
[0144] Table 8. Solubility of CPS at room temperature and 60°C
[0145] 3.8: In vitro antioxidant activity
[0146] The concentration-free radical scavenging rate graphs of five parallel experiments for HWE-CPS and UAE-CPS are shown below. Figure 5 As shown, the antioxidant activity of CPS is dose-dependent, and the antioxidant activity of UAE-CPS is superior to that of HWE-CPS.
[0147] 3.8.1: Assay of DPPH free radical scavenging activity
[0148] DPPH, with its stable nitrogen-centered free radical, is often used to evaluate the in vitro antioxidant properties of polysaccharides. When a single electron from the nitrogen atom of a DPPH free radical comes into contact with an electron-absorbing antioxidant, the DPPH ethanol solution will decolorize. The scavenging abilities of vitamin C and cytosolic acid (CPS) against DPPH free radicals at specific concentration gradients are shown in the figure. Figure 5 As shown in (a).
[0149] 3.8.2. Hydroxyl radical scavenging activity assay
[0150] Most biomolecules can react with hydroxyl radicals (a type of highly aggressive reactive oxygen species). Let's see how effectively they combat free radicals. Antioxidants provide hydrogen to bind with hydroxyl radicals, thus halting the free radical reaction chain and achieving an antioxidant effect. Figure 5 (b) It can be seen that CPS has a certain ability to scavenge hydroxyl radicals and has a certain antioxidant capacity.
[0151] 3.8.3: Assay of ABTS free radical scavenging activity
[0152] ABTS radicals are a common free radical used to evaluate the antioxidant capacity of natural polysaccharides. The hydroxyl and carboxyl groups of antioxidants can donate electrons to convert ABTS radicals into a stable form, thus exhibiting antioxidant activity. The scavenging rate of CPS increases with increasing solubility. Figure 5 As shown in (c), the scavenging rates of Vc and CPS increase with increasing concentration.
[0153] 3.8.4: Comparison of free radical scavenging rates between HWE-CPS and UAE-CPS
[0154] To clarify the difference in the effects of HWE and UAE on the antioxidant activity of CPS, the IC50 of three parallel experiments of HWE-CPS and UAE-CPS were calculated for t-test significance analysis.
[0155] (3.2)
[0156] In the formula: D1: defines a polysaccharide concentration below 50%; D2: defines a polysaccharide concentration above 50%; P1: free radical scavenging rate at concentration D1; P2: free radical scavenging rate at concentration D2;
[0157] Based on the calculations, and by performing a t-significance test on five parallel data sets of IC50, the results are shown in the table.
[0158] Known It refers to the concentration at which an active substance (such as polysaccharides, polyphenols, drugs, etc.) inhibits a specific biological process (such as free radical generation, enzyme activity, cell proliferation) by 50%. The smaller the value, the lower the concentration at which the sample achieves a 50% free radical scavenging rate, and the stronger the biological activity. Correspondingly, in this study… The smaller the value, the lower the concentration at which the polysaccharide achieves a 50% free radical scavenging rate, and the stronger its antioxidant activity. After a significant t-test, it was found that UAE-CPS exhibited higher scavenging rates for DPPH, ABTS+, and ·OH than HWE-CPS, with significant differences in scavenging rates for ABTS+ and ·OH. This indicates that UAE-CPS has significantly stronger antioxidant activity than HWE-CPS. The reason for this is that hot water extraction typically requires high temperatures (80-100℃) and prolonged heating. High temperatures easily lead to the breakage of glycosidic bonds in Codonopsis pilosula polysaccharides, causing long-chain polysaccharides to degrade into smaller molecular fragments and even destroying the branched structure of polysaccharides. Simultaneously, high temperatures promote the recombination of intramolecular and intermolecular hydrogen bonds in polysaccharides, resulting in the encapsulation of active groups (such as hydroxyl and carboxyl groups). In contrast, ultrasound-assisted enzyme extraction operates at lower temperatures (typically 40-60℃, the optimal temperature for the enzyme) and shorter extraction times, significantly reducing the risk of high-temperature degradation and structural rearrangement, thus better preserving the natural active structure of the polysaccharide. This results in better antioxidant activity.
[0159]
[0160] Table 9. Free radical scavenging rate IC50 significance analysis of CPS
[0161] 3.8 In vitro antibacterial activity
[0162] 4. Conclusion
[0163] This study employed ultrasound-assisted enzymatic extraction to extract polysaccharides (CPS) from Codonopsis pilosula. A composite enzyme of cellulase and papain at a ratio of 1:2 significantly improved the yield of CPS. RSM experiments were conducted to optimize the ultrasound-assisted enzymatic extraction process, yielding the ideal extraction parameters: a liquid-to-solid ratio of 20.49 mL / g, an ultrasound time of 40.49 min, an ultrasound power of 398.36 W, and an enzyme level of 2.53%. The expected ideal yield was 30.34%, significantly higher than that of HWE (20.07%). The sugar content of UAE-CPS (79.67%) was significantly higher than that of HWE-CPS (69.24%), while the protein content of UAE-CPS (6.94%) was significantly lower than that of HWE-CPS (8.87%). The zeta potential (+8.5 mV) of UAE-CPS was significantly higher than that of HWE-CPS (+6.52 mV), and the particle size of UAE-CPS (2186.2 nm) was significantly smaller than that of HWE-CPS (5473.42 nm), which is consistent with the SEM results. Correspondingly, the solubility of UAE-CPS (14.32%) was also significantly higher than that of HWE-CPS (11.42%). The determination of its antioxidant activity revealed that UAE-CPS exhibited significantly stronger antioxidant activity than HWE-CPS, suggesting its potential applications in the food and pharmaceutical industries. Furthermore, structural analysis of UAE-CPS showed that the extraction process used in this study did not damage the CPS structure. These data indicate that UAE is a better choice than HWE. However, the higher-order structure of CPS and its conformational relationships remain unclear and require further investigation.
Claims
1. A method for extracting highly active Codonopsis pilosula polysaccharides, characterized in that: The process includes raw material pretreatment, preparation of compound enzyme preparation, ultrasound-assisted enzyme extraction, and post-treatment of the extract. The ultrasound-assisted enzyme extraction and enzymatic hydrolysis processes are carried out simultaneously. The post-treatment includes centrifugation and filtration, vacuum concentration, alcohol precipitation purification, deproteinization, and freeze-drying.
2. The extraction method of highly active Codonopsis pilosula polysaccharides according to claim 1, characterized in that: The specific pretreatment of the raw materials involves: selecting Sichuan Codonopsis pilosula from Kaizhou, Chongqing, China, and then... After washing, freeze-dry, pulverize and pass through a 180-mesh sieve, then seal and store. The Codonopsis pilosula powder was mixed with petroleum ether at a ratio of 1:5, and extracted by reflux at 50°C for 2 hours to remove lipids and pigments. After drying, Codonopsis pilosula powder was obtained.
3. The extraction method of highly active Codonopsis pilosula polysaccharides according to claim 1, characterized in that: The compound enzyme preparation is made by uniformly mixing papain and cellulase at a mass ratio of 2:1, and then sealing it for later use.
4. The extraction method of highly active Codonopsis pilosula polysaccharides according to claim 1, characterized in that: The parameters for ultrasound-assisted enzyme extraction are: ultrasound frequency 40kHz, ultrasound power 398.36W, ultrasound time 40.49min, liquid-to-solid ratio of extraction system is 20.49mL / g, and the amount of compound enzyme preparation added is 2.53% of the mass of Codonopsis pilosula powder.
5. The extraction method of highly active Codonopsis pilosula polysaccharides according to claim 1, characterized in that: The alcohol precipitation purification process involves adding 4 times the amount of anhydrous ethanol to the concentrate, allowing it to stand at 4°C for 12 hours, and then centrifuging to separate the precipitate.
6. The extraction method of highly active Codonopsis pilosula polysaccharides according to claim 1, characterized in that: The deproteinization process involves mixing polysaccharide solution, chloroform, and n-butanol in a volume ratio of 15:4:
1. After thorough mixing, the bottom protein layer and organic layer are removed. This process is repeated until the protein is completely removed.
7. The extraction method of highly active Codonopsis pilosula polysaccharides according to claim 1, characterized in that: The freeze-drying time was 48 hours, and the yield of the prepared Codonopsis pilosula polysaccharide product was 30.34%. The polysaccharide content of the Codonopsis pilosula polysaccharide was 79.67%, the protein content was 6.94%, the uronic acid content was 0.011%, and the total phenol content was 0.010%.
8. The extraction method of highly active Codonopsis pilosula polysaccharides according to claim 1, characterized in that: The centrifugation parameters after centrifugation filtration and alcohol precipitation purification are: centrifugation speed 5000 rpm, centrifugation time 5 min.
9. The extraction method of highly active Codonopsis pilosula polysaccharides according to claim 1, characterized in that: During the ultrasound-assisted enzyme extraction, distilled water was used as the solvent in the extraction system. After extraction, the mixture was cooled to room temperature before centrifugation and filtration.
10. The method for extracting highly active Codonopsis pilosula polysaccharides according to claim 1, characterized in that: In the raw material pretreatment, Codonopsis pilosula is freeze-dried and then sealed for storage, and then extracted with petroleum ether by reflux and dried for later use, to ensure that the Codonopsis pilosula powder is free of lipid and pigment residues.