Method for extracting active polysaccharide from red paeony root by using eutectic solvent

Abstract

The invention provides a method for extracting active polysaccharides from red peony roots by using a deep eutectic solvent. When the deep-eutectic solvent, especially the deep-eutectic solvent composed of choline chloride and citric acid, is used for extracting the radix paeoniae rubra active polysaccharide, the extraction rate of the polysaccharide is high, the prepared radix paeoniae rubra active polysaccharide has a strong nerve cell protection effect, and the deep-eutectic solvent can be repeatedly used for multiple times. Therefore, the method disclosed by the invention is simple and easy to implement and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to a method for extracting active polysaccharides from Paeonia lactiflora using a eutectic solvent. Background Technology

[0002] Red peony root, the dried root of Paeonia lactiflora Pall. or Paeonia veitchii Lynch, belonging to the Ranunculaceae family, has a medicinal history of over two thousand years and possesses high medicinal value. Modern research indicates that red peony root has a complex chemical composition, rich in glycosides, phenolic acids, flavonoids, and polysaccharides, and exhibits various effects such as anti-inflammatory, antioxidant, anti-tumor, and hypoglycemic properties.

[0003] Different solvents or methods used to extract Paeonia lactiflora result in extracts containing different active ingredients, thus exhibiting varying pharmaceutical activities. Chinese patent CN118852477A discloses a method that combines petroleum ether defatting, water extraction with alcohol precipitation, and an enzyme-Sevag method, along with DEAE-52 column chromatography to prepare high-purity Paeonia lactiflora polysaccharide RPP-1 for diabetes treatment. However, this method is cumbersome and difficult to scale up for industrial production.

[0004] Parkinson's disease (Parkinson's disease) is the second most common neurodegenerative disease, posing a significant threat to human health. The main pathological features of Parkinson's include the progressive degeneration of dopaminergic neurons in the substantia nigra compacta and the formation of Lewy bodies. Currently, there is no cure or treatment to stop the progression of Parkinson's disease. Its pathogenesis involves multiple interacting pathways, including oxidative stress, neuroinflammation, mitochondrial dysfunction, apoptosis, and gut-brain axis disorders. Single-target drugs often fail to effectively halt disease progression. This complex pathological network supports the theoretical basis for multi-component, multi-target intervention strategies in traditional Chinese medicine. Modern pharmacological studies have found that Paeonia lactiflora (red peony root) and its compound traditional Chinese medicine formulas can exert neuroprotective effects by inhibiting oxidative stress, reducing neuroinflammation, and promoting autophagy. Human neuroblastoma (SH-SY5Y) is a dopaminergic neuronal cell line that can directly mimic neurons and is frequently used as an in vitro model for neuronal cell experiments, a method widely accepted in the academic community for studying neuronal damage. Modern pharmacological studies have shown that Paeonia lactiflora can reduce H2O2-induced SH-SY5Y cell damage and apoptosis.

[0005] In summary, this study proposes a novel and effective method for extracting Paeonia lactiflora polysaccharides, which is characterized by high efficiency, simplicity, and good efficacy of the extracted Paeonia lactiflora polysaccharides. Summary of the Invention

[0006] To address the problems of existing technologies, the purpose of this invention is to design a technical solution and provide a method for extracting active polysaccharides from Paeonia lactiflora.

[0007] This invention is specifically achieved through the following technical solutions: A method for extracting active polysaccharides from Paeonia lactiflora using a eutectic solvent, the method comprising: heating and extracting Paeonia lactiflora powder in a eutectic solvent, collecting the supernatant by centrifugation, precipitating with alcohol, centrifuging again, and freeze-drying the precipitate to obtain active polysaccharides.

[0008] Furthermore, the eutectic solvent is composed of hydrogen bond acceptors HBAs and hydrogen bond donors HBDs, wherein the hydrogen bond acceptors HBAs are selected from at least one of choline chloride and betaine; and the hydrogen bond donors HBDs are selected from at least one of 1,4-butanediol, glycerol, ethylene glycol, malic acid, DL-lactic acid, citric acid, 1,2-propanediol, and urea. The water content of the eutectic solvent is 0.3-0.4 g / g eutectic solvent.

[0009] Furthermore, the method specifically includes the following steps: Preparation of eutectic solvent: Hydrogen bond acceptors HBAs and hydrogen bond donors HBDs are mixed and heated under stirring until they melt until a clear and homogeneous solvent is obtained. The heating temperature can be 60-80°C, specifically 60°C, 70°C, or 80°C, and the time can be 1-2 hours, specifically 1 hour or 2 hours.

[0010] Furthermore, the molar ratio of the hydrogen bond acceptors HBAs to the hydrogen bond donors HBDs can be 1:2-3:1, specifically 1:2, 2:2, 5:4, 3:2, 2:1, or 3:1.

[0011] Furthermore, the hydrogen bond acceptor is choline chloride, the hydrogen bond donor is citric acid, and the molar ratio of choline chloride to citric acid is 1:2-2:1, preferably 1:2, 2:2, 5:4, 3:2, 2:1, and more preferably 3:2.

[0012] Furthermore, in this method, the ratio of peony powder to eutectic solvent can be 1 mg: 10-50 mL, specifically: 1 mg: 10 mL, 1 mg: 20 mL, 1 mg: 30 mL, 1 mg: 40 mL, 1 mg: 50 mL.

[0013] Furthermore, the extraction temperature can be 40-80℃, specifically 40℃, 50℃, 60℃, 70℃, or 80℃, and the extraction time can be 20-70 min, specifically 45 min, 50 min, 60 min, or 70 min.

[0014] Furthermore, the water content of the eutectic solvent is 0.4 g / g eutectic solvent.

[0015] Furthermore, the centrifugation conditions for collecting the supernatant are 12000-15000 rpm for 10-25 min, specifically 13000 rpm for 20 min.

[0016] Furthermore, the alcohol precipitation uses ethanol with a volume concentration of 75%-95%, specifically ethanol with a volume concentration of 95%.

[0017] The volume of ethanol used for alcohol precipitation is 1-5 times the volume of the supernatant, specifically 1, 2, 3, 4, or 5 times.

[0018] Furthermore, the alcohol precipitation temperature can be 0~4℃, and the time can be 6 to 24 hours.

[0019] Furthermore, the conditions for centrifugation after alcohol precipitation are 5000-8000 rpm for 10-25 min, specifically 8000 rpm for 10 min.

[0020] The active polysaccharide of Paeonia lactiflora obtained by the above method is also within the scope of protection of this invention.

[0021] The present invention also provides the application of the aforementioned active polysaccharide of Paeonia lactiflora in the preparation of drugs that promote the proliferation of SH-SY5Y cells.

[0022] The present invention also provides the application of the aforementioned active polysaccharide of Paeonia lactiflora in the preparation of neuroprotective drugs.

[0023] Furthermore, the neuroprotective drugs are used to prevent or treat Parkinson's disease and Alzheimer's disease.

[0024] Compared with the prior art, the present invention has the following advantages: This invention provides a method for extracting active polysaccharides from Paeonia lactiflora using a eutectic solvent. The invention discovers that using a eutectic solvent, particularly one composed of choline chloride and citric acid, results in a high extraction rate of the polysaccharides. Furthermore, the extracted active polysaccharides exhibit excellent promotion of SH-SY5Y cell proliferation and demonstrate good neuroprotective effects. Simultaneously, the eutectic solvent can be easily and repeatedly reused, maintaining a higher extraction rate than water extraction. This method is simple and easy to implement, and has promising application prospects. Attached Figure Description

[0025] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1The standard curve of polysaccharides in Example 1 of this invention (A), and the polysaccharide extraction rates with different NADES (B); one-way ANOVA, n=3. .

[0026] Figure 2 The effects of polysaccharides extracted from different NADES on the viability of SH-SY5Y cells in Example 1 of this invention (A); the effects of polysaccharides extracted from different NADES on the viability of MPP+-induced SH-SY5Y cells (B); one-way ANOVA, n=3, .

[0027] Figure 3 The following images show the effects of different moisture contents of NADES on the polysaccharide extraction rate in Example 1 of this invention (A); infrared spectrum of choline chloride (B); infrared spectrum of citric acid (C); and infrared spectrum of NADES-6 (D).

[0028] Figure 4 The effect of the molar ratio of solvent to polysaccharide on polysaccharide extraction rate (A); the effect of extraction time on polysaccharide extraction rate (B); the effect of extraction time on polysaccharide extraction rate (C); the effect of solid-liquid ratio on polysaccharide extraction rate (D).

[0029] Figure 5 shows the response surface plot and contour plot (A) of the interaction of various factors affecting polysaccharide extraction rate; and the response surface plot and contour plot (B) of the interaction of various factors affecting cell viability.

[0030] In Figure 6, (A) shows the effect of the number of extractions on the polysaccharide extraction rate; (B) shows the recovery rate of NADES-6. Detailed Implementation

[0031] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0032] The choline chloride, 1,4-butanediol, glycerol, ethylene glycol, malic acid, DL-lactic acid, 1,2-propanediol, urea, betaine, glucose, and levodopa used in the following examples were all purchased from Haialadin Biochemical Technology Co., Ltd., Nile Red was purchased from Shanghai Jizhi Biochemical Technology Co., Ltd., MPP+ was purchased from Sigma, and 25T culture flasks were purchased from NEST. Example 1: Investigation of the composition and water content of the eutectic solvent 1. Sample preparation: Paeonia lactiflora Pall. or Paeonia veitchii Lynch was harvested in Daxiling City, Heilongjiang Province, and purchased in November 2024. The dried Paeonia lactiflora slices were pulverized and passed through a 60-mesh sieve to obtain Paeonia lactiflora powder for later use.

[0033] 2. Solvent determination: Natural eutectic solvents (NADES) were synthesized by mixing choline chloride, betaine, and other HBDs in a specific molar ratio, heating in a water bath at 80°C, and magnetically stirring for 2 hours until a clear and homogeneous solvent was obtained. Since solvents generally have high viscosity, which is detrimental to polysaccharide dissolution and mass transfer, and makes extraction and separation difficult, 30 wt% distilled water was added during the initial screening to accelerate the synthesis while reducing the system viscosity. After synthesis, the NADES were stored at room temperature. The different types of NADES used in this experiment, their numbers, compositions, and extraction conditions are shown in Table 1.

[0034] Table 1. Molar ratios and numbering of different NADES 3. Polysaccharide content determination and extraction rate calculation: The phenol-sulfuric acid method is a widely used method for determining polysaccharides, exhibiting high sensitivity and simple operation, suitable for the determination of most polysaccharides. 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of 0.1 mg / mL standard glucose solution were respectively measured into stoppered colorimetric tubes, and then brought to a final volume of 2 mL with deionized water to obtain glucose standard solutions of different concentrations. 1 mL of 6% phenol solution and 5 mL of concentrated sulfuric acid were added to each of the above eight colorimetric tubes, and the mixture was vortexed on a vortex apparatus. After boiling in a water bath for 10 min and cooling in ice water, the absorbance was measured at 490 nm. The data were processed using GraphPad 10 software, with glucose concentration as the x-axis and absorbance as the y-axis, and a standard curve was plotted. The polysaccharide content in the sample was calculated based on the glucose standard curve.

[0035] The formula for calculating the extraction rate of Paeonia lactiflora polysaccharides is as follows: C: Polysaccharide concentration calculated from the glucose standard curve, mg / mL; V: Volume of polysaccharide after final volume adjustment, mL; N: Dilution factor; M0: Mass of Paeonia lactiflora powder, g; A glucose standard curve was established using the phenol-sulfuric acid method, and the regression equation obtained was: Y = 0.1315X + 0.002937, R² = 0.9959. Figure 1 (A)

[0036] 4. Solvent determination (1) Polysaccharide extraction rate Accurately weigh 0.5 g of Paeonia lactiflora powder and place it in an Erlenmeyer flask. Add pre-prepared NADES, stir well, let stand for 5 min, and then extract at 60℃ for 30 min. Centrifuge the solution at 13000 rpm for 20 min, collect the supernatant, add 4 volumes of 95% ethanol, precipitate overnight at 4°C, centrifuge at 8000 rpm for 10 min to obtain crude polysaccharide, freeze-dry, and prepare a 0.1 mg / mL solution using 1 mg of the sample. Calculate the polysaccharide content and extraction rate, and select the solvent with the highest polysaccharide extraction rate for subsequent experiments. Simultaneously, hot water extraction of Paeonia lactiflora polysaccharide was used as a blank control to evaluate the efficiency of NADES extraction. Centrifuge at 8000 rpm for 10 min in a boiling water bath with the same liquid-to-solid ratio for 3 h. Precipitate with 4 volumes of 95% ethanol, centrifuge, collect the supernatant, and incubate overnight at 4℃. The resulting precipitate was washed twice with ethanol, dissolved, reconstituted with ultrapure water, and freeze-dried to obtain NADES and polysaccharides extracted by conventional methods.

[0037] Figure 1 B represents the polysaccharide extraction rate for different NADES values; one-way ANOVA, n=3. .

[0038] NADES, as the extraction solvent, has a significant impact on the extraction rate of PRRP. The results show that polyacid-based solvents have a higher extraction rate of polysaccharides compared to polyol-based solvents, while the polysaccharide extraction rate of polyol-based solvents is lower than that of water extraction. Among them, the betaine-urea type solvent has the highest polysaccharide extraction rate (17.16%), followed by the choline chloride-citric acid type solvent (14.31%).

[0039] (2) Cell viability assay SH-SY5Y cells were purchased from a Korean cell bank and cultured in DEME, penicillin-streptomycin-amphoteric acid B, and other environments. SH-SY5Y cells were cultured in 10% FBS at 37°C in a 5% CO2 incubator. SH-SY5Y cells were seeded at a density of cells / well in 96-well plates. After culturing SH-SY5Y cells for 24 h, NADES-extracted polysaccharides were prepared in complete culture medium at a concentration of 500 μg / mL. 100 μL of the solution was added to each cell well. After culturing for 22 h, 10 μL of LCK8 was added to each well. The viability of SH-SY5Y cells in each well was measured at 450 nm to evaluate the cytotoxicity of NADES-extracted polysaccharides to SH-SY5Y cells.

[0040] Subsequently, SH-SY5Y nerve cells were used... SH-SY5Y cells were seeded at a density of cells / well in 96-well plates. After 24 h of seeding, except for the blank control and negative control groups, 100 μL of MPP+ (prepared to a 1 mM solution with basal medium) was added to each well to treat SH-SY5Y cells for 24 h. Then, NADES-extracted polysaccharides (500 μg / mL) were added and cultured for another 22 h. Finally, 10 μL of LCK8 was added to each well, and the viability of SH-SY5Y cells in each well was measured at 450 nm to evaluate the effect of NADES-extracted polysaccharides on MPP+-induced SH-SY5Y PD cell viability.

[0041] To further determine the effect of NADES on polysaccharide extraction, cell experiments were conducted to determine the effect of 12 NADES extracts on the cell viability of SH-SY5Y cells. The cytotoxicity of NADES-extracted polysaccharides was evaluated at different doses. NADES-extracted polysaccharides showed no cytotoxicity at 500 μg / mL, therefore, 500 μg / mL was selected for the next step of cell viability testing (Figure 2A). The effect of NADES-extracted polysaccharides on SH-SY5Y cell viability was further screened. An SH-SY5Y PD cell model was established by culturing cells with 1 mM MPP+. NADES-extracted polysaccharides increased the MPP+-induced cell viability of SH-SY5Y cells (p<0.01). Among them, NADES-6-extracted polysaccharides showed the highest SH-SY5Y cell viability (93.55%), followed by NADES-8 (91.82%). Based on the conditions of high polysaccharide extraction rate and promotion of SH-SY5Y PD cell proliferation, this invention selected NADES-6 as the extraction solvent for subsequent experimental studies. Figure 2 (B)

[0042] 5. Determination of moisture content Due to its tight hydrogen bond network structure, NADES often exhibits high viscosity and poor flowability. While increasing hydrogen bonds can improve solubility, high viscosity is detrimental to mass transfer during extraction. Introducing water into NADES is a common method to reduce viscosity, promote mass transfer, and improve extraction performance. However, the physical properties of the solution (such as pH, density, polarity, and surface tension) change with the addition of water, which also affects extraction efficiency.

[0043] This invention evaluates the yield of polysaccharides by adding different water contents (20%, 30%, 40%, 50%, 60%, 70%) to obtain the optimal NADES-6, in order to select the optimal water content.

[0044] The extraction efficiency of NADES is affected by its water content, making it a crucial factor influencing polysaccharide extraction rate. Strong hydrogen bonds between components are disrupted with increasing water content, thus impacting polysaccharide extraction. Extensive hydrogen bonds form between solvent components, but these bonds gradually weaken with increasing water content; beyond a certain level, the hydrogen bonding effect may be severely weakened or even disappear. Furthermore, water content significantly affects solvent viscosity. As water content increases, the viscosity of the solvent system decreases, which facilitates the complete dissolution and mass transfer of polysaccharides, thereby improving the extraction rate. The polysaccharide extraction rate significantly increases from 20% to 40% water content. However, a further increase from 40% to 70% significantly reduces the extraction rate. Therefore, this study selected a solvent water content of 40% (Figure 3A).

[0045] Hydrogen bonds consist of hydrogen bond donors and hydrogen bond acceptors, and the formation of NADES is also due to the ability of these two to form a complex hydrogen bond network structure. Therefore, infrared spectroscopy will be used next to verify whether the preparation of NADES-6 was successful.

[0046] 6. Structural characterization of NADES-6 Fourier transform infrared spectroscopy was used to measure the characteristic infrared absorption peaks of choline chloride, citric acid, and their eutectic solvent NADES-6, respectively. The changes in the characteristic infrared absorption peaks of the three were compared to determine whether hydrogen bonds formed between choline chloride and citric acid. Appropriate amounts of NADES-6, choline chloride, and citric acid were thoroughly ground with potassium bromide, and then scanned using a Fourier transform infrared spectrometer in the range of 4000–500 cm⁻¹.

[0047] 7. Determination of the physicochemical properties of NADES-6 (1) pH measurement To measure the pH of the sample using a pH meter, first calibrate the electrode by inserting it into calibration buffers at pH 4.00 and pH 6.86. After calibration, immerse the electrode 3-5 cm below the surface of the sample solution and measure the pH of the polysaccharide. Repeat the test three times for each sample.

[0048] (2) Density measurement The density of NADES-6 was measured at room temperature using a laboratory densitometer.

[0049] (3) Polarity measurement Since the maximum absorption wavelength of Nile red dye in the UV-Vis region is easily affected by solvent polarity, the polarity of NADES-6 can be evaluated based on the molar transition energy (ENR) of the probe dye in the solvent. Weigh 1 mg of Nile red (NR) and add it to 1 mL of anhydrous ethanol to prepare a 1 mg / mL Nile red solution, which is stored at 4°C protected from light. Add 0.8 mL of NADES-6 to 0.1 mL of the Nile red solution, and perform a wavelength scan in the range of 200-800 nm⁻¹, recording the maximum absorption wavelength. Calculate the ENR (kJ / mol) using the following formula (1-2) to represent the polarity.

[0050] h: Planck's constant; c: speed of light; NA: Avogadro's constant; λmax: maximum absorption wavelength.

[0051] (4) Viscosity measurement The viscosity of NADES-6 was determined using a Thermo HAAKE MARS 60 instrument equipped with a 27 mm diameter double-slit coaxial cylindrical rotor. Approximately 3.5 mL of solution was loaded at 25°C. The sample was held at this temperature for 90 s before testing. Then, within 120 s, the shear rate was measured by scanning a linear distribution from 0.1 to 500 s⁻¹, with 100 data points collected. Each sample was measured three times.

[0052] (5) Surface tension measurement The surface tension of NADES-6 was measured using a Theta Flex contact angle analyzer.

[0053] This invention comprehensively determined the physical properties of a 40% choline chloride-citric acid solution using the aforementioned method. Table 2 shows that the addition of water reduced the density and viscosity of NADES-6, promoting mass transfer during the extraction process. NADES is a high-viscosity solvent, but the addition of 40% water significantly reduced the viscosity of NADES-6, placing it below the viscosity threshold (100 mPa / s) of traditional industrial solvents. The surface tension of NADES is increased due to the presence of hydrogen bonds, resulting in stronger intermolecular forces. With increasing water content, the surface tension of NADES begins to decrease, intermolecular forces weaken, and viscosity decreases. However, the addition of large amounts of water can also disrupt the structure of NADES to some extent, while small amounts can improve its stability. ENR is widely used to indicate the polarity of NADES; a smaller ENR value indicates greater polarity. The table shows that the polarity of NADES-6 is 124.67. Furthermore, its pH indicates that it is a strongly acidic solution.

[0054] Table 2. Physical properties of NADES-6 Example 2: Single-factor experimental design (1) Molar ratio Accurately weigh 0.5 g of Paeonia lactiflora powder, fix the extraction temperature at 60°C, the extraction time at 45 min, and the material-to-liquid ratio at 1:30. Using the polysaccharide extraction rate as the indicator, investigate the effect of the molar ratio of choline chloride to citric acid (1:1, 1:2, 1:3, 2:1, 3:1) on the polysaccharide extraction rate.

[0055] The molar ratio of choline chloride to citric acid in the solvent directly determines the strength of hydrogen bonding interactions between solvents, thus affecting the solubility and extraction efficiency of polysaccharides. When the molar ratio of citric acid in the solvent increases from 1:1 to 1:3, the polysaccharide extraction rate increases significantly; however, when the molar ratio of choline chloride increases from 1:1 to 3:1, the polysaccharide extraction rate does not increase and even decreases. Furthermore, the viscosity of the solvent increases, which may adversely affect the solubility and mass transfer of polysaccharides. The highest polysaccharide extraction rate is achieved at a solvent molar ratio of 1:3; therefore, a solvent molar ratio of 1:3 was chosen for subsequent experiments. Figure 4 (A)

[0056] (2) Extraction temperature Accurately weigh 0.5 g of Paeonia lactiflora powder, fix the molar ratio at 1:3, the extraction time at 45 min, and the material-liquid ratio at 1:30. Using the polysaccharide extraction rate as the indicator, investigate the effect of extraction temperature (40°C, 50°C, 60°C, 70°C, 80°C) on the polysaccharide extraction rate.

[0057] Temperature is one of the key factors affecting polysaccharide extraction. Generally, increased temperature increases the solubility of polysaccharides to some extent because higher temperatures weaken intermolecular forces, making polysaccharide molecules easier to dissolve. It is worth noting that temperature has a drastic effect on reducing solvent viscosity; increased temperature may disrupt the eutectic phase, leading to a looser solvent structure and reduced viscosity. When the extraction temperature increased from 40℃ to 60℃, the polysaccharide extraction rate gradually increased. However, when the temperature reached 70℃-80℃, the extraction rate decreased significantly, possibly due to excessively high temperatures causing structural changes or even denaturation of the polysaccharides. Therefore, an extraction temperature of 60℃ was chosen for subsequent experiments (Figure 4B).

[0058] (3) Extraction time Accurately weigh 0.5 g of Paeonia lactiflora powder, fix the extraction temperature at 60°C, the molar ratio at 1:3, and the material-liquid ratio at 1:30, and investigate the effect of extraction time (15 min, 30 min, 45 min, 60 min, 75 min) on the polysaccharide extraction rate, using the polysaccharide extraction rate as the indicator.

[0059] The extraction time directly affects the contact time and mass transfer between the solvent and the medicinal material components during extraction, thus influencing the dissolution and extraction efficiency of polysaccharides. Some polysaccharide extraction processes may require a longer extraction time to reach mass transfer equilibrium, but excessively long processing times may alter the polysaccharide structure, thereby reducing the extraction rate and causing unnecessary waste of raw materials and energy. Within the first 60 minutes of extraction, the polysaccharide extraction rate significantly increased with increasing extraction time, but decreased slightly at 75 minutes, possibly indicating the reaching of an equilibrium between polysaccharide dissolution and accumulation and partial degradation. Therefore, an extraction time range of 60 minutes was selected for subsequent experiments (Figure 4C).

[0060] (4) Liquid-to-solid ratio Accurately weigh 0.5 g of Paeonia lactiflora powder, fix the extraction time at 45 min, the extraction temperature at 60°C, and the molar ratio at 1:3. Using the polysaccharide extraction rate as the indicator, investigate the effect of the material-liquid ratio (1:10, 1:20, 1:30, 1:40, 1:50) on the polysaccharide extraction rate.

[0061] The solid-liquid ratio directly affects the contact between the solvent and the raw material during polysaccharide extraction and the extraction efficiency of polysaccharides. A higher solid-liquid ratio allows more solvent to wet the raw material, resulting in more effective release and dissolution of polysaccharides in the solvent, accelerating the mass transfer rate, and thus improving extraction efficiency. On the other hand, an excessively high solid-liquid ratio may lead to saturation of polysaccharide dissolution in the solvent, ceasing to promote mass transfer, and also causing solvent waste and increasing costs. The results show that when the solid-liquid ratio increases from 1:10 to 1:40, the polysaccharide extraction rate increases significantly. When the solid-liquid ratio is further increased to 1:50, the polysaccharide extraction rate tends to stabilize. Therefore, a solid-liquid ratio of 1:40 was selected for subsequent experiments (Figure 4D).

[0062] (5) Response surface experimental design and model establishment The Box-Behnken experimental design of Response Surface Methodology (RSM) is widely used for extraction optimization because it can fit the optimal process conditions with a small number of experimental groups. Based on the results of single-factor experiments, four independent variables (A is the molar ratio, B is the extraction temperature, C is the extraction time, and D is the solid-liquid ratio) were determined, and the polysaccharide extraction rate was used as the response value, i.e., the dependent variable. Thus, the optimal combination of the four variables was obtained, and the theoretical optimal extraction process conditions were obtained. 29 sets of experiments were designed using Design-Expert 13 software, with four variables and three levels for each variable. The experimental design results are shown in Table 4.

[0063] Table 3. Factors and Levels in Response Surface Analysis The significance of the model was evaluated using analysis of variance (ANOVA), with formulas 1-3 representing the response variables. Y The equation that determines the correlation between variables: Y : Extraction rate of polysaccharides; Xi or j (i or j = 1, 2, 3, 4): Four variables; 0: Constant term; i (i = 1,2,3,4): coefficients of the linear part. ij (i,j =1,2,3): coefficients of the quadratic term, ε: residuals. The goodness of fit of the model equations is measured by the coefficient of determination (R²). 2 The F-test was used to determine statistical significance and the significance of regression coefficients. P <0.05 indicates significance.

[0064] Based on the results of the single-factor experiments, the solvent molar ratio, extraction temperature, extraction time and solid-liquid ratio for extracting PRRP polysaccharides from NADES-6 were optimized using the Box-Behnken experimental design. The experimental design and results are shown in Table 4.

[0065] Table 4 Results of the Box-Behnken Experimental Design (1) Model establishment and statistical analysis Design Expert software, through multivariate nonlinear regression fitting of the data, obtained the following multivariate quadratic regression equation for the polysaccharide extraction rate based on molar ratio, extraction time, extraction temperature, and material-liquid ratio: Box-Behnken analysis showed that the experimental model had an F-value of 146.12, with a limiting significance (p<0.0001), and no significant lack-of-fit term (p>0.05). The R², corrected R², and predicted R² values ​​were 0.9932, 0.9864, and 0.9776, respectively, with a CV% of 1.16% < 10%, indicating that the constructed model had good fit and could be used for analysis and prediction. Furthermore, extraction temperature, extraction time, and the solid-liquid ratio (p<0.01) significantly affected the polysaccharide extraction efficiency. The interaction terms AB, BC, CD, A², B², C², and D² of each factor had significant effects on the polysaccharide extraction rate (p<0.05, p<0.01). Based on the F-value, the factors with the greatest impact on the polysaccharide extraction rate were the solid-liquid ratio and extraction time, followed by extraction temperature, while the molar ratio had a smaller impact (Table 5).

[0066] Table 5. Results of ANOVA for the Polysaccharide Extraction Rate Regression Model .

[0067] 3D response surface methodology (RSM) can more intuitively display the complex relationships between multiple variables, while two-dimensional contour plots can simplify the data and highlight the main trends, reflecting the significance of the interaction between two factors. The more elliptical the ellipse, the more significant the interaction between the two factors. The greater the slope of the curve in the 3D RSM, the greater the influence of that factor on the extraction result. As shown in Figure 5A, the interaction between solvent molar ratio and extraction temperature, extraction temperature and extraction time, and extraction time and solid-liquid ratio have a significant impact on the polysaccharide extraction rate (p<0.05). The contour plots of solvent molar ratio and extraction time, molar ratio and solid-liquid ratio, and extraction temperature and solid-liquid ratio are close to circular, and their interaction effects on the polysaccharide extraction rate are not significant (p>0.05), which is consistent with the variance and significance analysis results in Table 6. The optimal extraction process obtained through software analysis is: molar ratio 1.28, extraction temperature 59.55°C, extraction time 74.44 minutes, and solid-liquid ratio 30.65. Under the above conditions, the predicted total extraction yield is 17.55%.

[0068] Table 6. Results of ANOVA for the Cell Viability Regression Model .

[0069] Meanwhile, the effects of molar ratio, extraction time, extraction temperature, and solid-liquid ratio on the viability of extracted polysaccharides induced by MPP+ were analyzed using Design Expert software. The multiple quadratic regression equation is as follows: Box-Behnken analysis showed that the experimental model had an F-value of 37.74, with p-limiting significance (p<0.0001), and no significant lack-of-fit term (p>0.05). The R², corrected R², and predicted R² values ​​were 0.9742, 0.9484, and 0.8954, respectively, with a CV% of 0.10% < 10%, indicating that the constructed model had good fit and could be used for analysis and prediction. Furthermore, the molar ratio (p<0.01) significantly affected the polysaccharide extraction efficiency. The interaction terms AD, BC, CD, A2, B2, C2, and D2 of each factor had significant effects on cell viability (p<0.05, p<0.01). Based on the F-value, the molar ratio had the greatest impact on cell viability, followed by the solid-liquid ratio, while extraction temperature and extraction time had relatively small effects.

[0070] Meanwhile, as shown in contour plot B of Figure 5, the order of influence of the four factors is MR > ET > ED > SSR. Furthermore, the order of influence of the interaction among the four factors on cell viability is CD > BC > BD > AD > AC. This aligns with the variance and significance analysis results and the 3D response surface methodology in Table 6. The 3D response surface plot shows that extraction time and material-liquid ratio significantly affect cell viability. However, higher molar ratio, material-liquid ratio, extraction time, and extraction temperature are not always better; therefore, optimal extraction conditions need to be selected. Software analysis yielded the following optimal extraction process: molar ratio 1.12, extraction temperature 60.36°C, extraction time 59.86 seconds, and material-liquid ratio 39.94. Under these conditions, the predicted polysaccharide activity is 89.81%.

[0071] Finally, combining the polysaccharide extraction rate and cell viability, and through software analysis, the optimal extraction conditions were determined to be: molar ratio 1.53, extraction temperature 60.49°C, extraction time 63.75 minutes, and solid-liquid ratio 36.31. The predicted polysaccharide extraction rate was 16.81%, and the cell viability was 88.20%.

[0072] In summary, based on actual experiments, the present invention selects a molar ratio of 1.5 (3:2), an extraction temperature of 60℃, an extraction time of 64 min, and a solid-liquid ratio of 36 mL / mg as the optimal extraction conditions.

[0073] (2) Optimal process verification Response surface methodology (RSM) optimization results showed that the optimal extraction conditions for PRRP in the ChCl-CA eutectic solvent system were a molar ratio of 1.5 (3:2), an extraction temperature of 60℃, an extraction time of 64 min, and a solid-liquid ratio of 36 mL / mg. Under these conditions, the predicted extraction rate was 16.81%, and the cell viability was 88.20%. Three validation experiments were conducted under these conditions, yielding a PRRP yield of 16.74% and a cell viability of 86.86%, which were not significantly different from the expected results in the model. This indicates that the optimization results are reliable and can be used for PRRP extraction (Table 7).

[0074] Table 7. Results of Optimal Process Validation Example 3: Investigating the performance of repeated solvent extraction The crude polysaccharide extract extracted according to the optimal extraction process was added to 95% ethanol and allowed to precipitate overnight. After centrifugation, the ethanol was collected and concentrated to 1 / 5 of the original volume by rotary evaporation. The solvent was recovered, and the extraction was repeated 6 times. The polysaccharide extraction rate was calculated, and the repeatability of the solvent was evaluated.

[0075] As shown in Figure 6, after six repeated extractions of the solvent, the polysaccharide extraction rate of PRRP decreased significantly, and the solvent recovery rate also decreased significantly. After four repeated extractions, the PRRP extraction rate remained above 10%, with the final extraction rate decreasing to 9.22%, but still higher than that of water extraction under the same conditions (8.78%). These studies indicate that NADES-6 possesses certain repeatability in extraction. The decrease in extraction rate may be related to the reduction in NADES-6 content in the solution. This reduction leads to a decrease in hydrogen bonds, and corresponding changes in physical properties also affect the extraction rate.

[0076] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for extracting active polysaccharides from Paeonia lactiflora, characterized in that, The method includes: heating and extracting Paeonia lactiflora powder in a eutectic solvent, collecting the supernatant by centrifugation, precipitating with alcohol, centrifuging again, and freeze-drying the precipitate to obtain active polysaccharides.

2. The method as described in claim 1, characterized in that, The eutectic solvent is composed of hydrogen bond acceptors HBAs and hydrogen bond donors HBDs. The hydrogen bond acceptors HBAs are selected from at least one of choline chloride and betaine. The hydrogen bond donors HBDs are selected from at least one of 1,4-butanediol, glycerol, ethylene glycol, malic acid, DL-lactic acid, citric acid, 1,2-propanediol, and urea. The water content of the eutectic solvent is 0.3-0.4 g / g eutectic solvent.

3. The method according to claim 2, characterized in that, The method includes: preparation of eutectic solvent: mixing hydrogen bond acceptors HBAs and hydrogen bond donors HBDs, heating them under stirring until a clear and homogeneous solvent is obtained, and then adding water and stirring evenly. The heating temperature is 60-80°C, and the time is 1-2 hours.

4. The method as described in claim 2, characterized in that, The molar ratio of hydrogen bond acceptors HBAs to hydrogen bond donors HBDs is 1:2-3:

1.

5. The method as described in claim 2, characterized in that, The hydrogen bond acceptor is choline chloride, the hydrogen bond donor is citric acid, and the molar ratio of choline chloride to citric acid is 1:2-2:

1.

6. The method as described in claim 1, characterized in that, In the method described, the ratio of peony powder to eutectic solvent is 1 mg: 10-50 mL.

7. The method as described in claim 1, characterized in that, The extraction temperature is 40-80℃, and the extraction time is 20-70 minutes.

8. The method as described in claim 1, characterized in that, The centrifugation conditions for collecting the supernatant are 12000-15000 rpm for 10-25 min. The alcohol precipitation uses ethanol with a volume concentration of 75%-95%; The volume of ethanol used for the alcohol precipitation is 1-5 times the volume of the supernatant; The alcohol precipitation is carried out at a temperature of 0-4°C for 6 to 24 hours. The conditions for centrifugation after alcohol precipitation are 5000-8000 rpm for 10-25 min.

9. The active polysaccharide of Paeonia lactiflora prepared by any one of claims 1-8.

10. The use of the active polysaccharide of Paeonia lactiflora according to claim 9 in the preparation of a drug that promotes the proliferation of SH-SY5Y cells.

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