Method for extracting effective components from radix foetidus by using a eutectic solvent system

Abstract

This invention discloses a method for extracting the effective components of Polygonum multiflorum based on a eutectic solvent system. The method involves dissolving Polygonum multiflorum powder in a eutectic solvent and then extracting it using ultrasound to obtain the effective components. The eutectic solvent includes hydrogen bond donor compounds and hydrogen bond acceptor compounds. The hydrogen bond donor compounds include one or more of xylitol, sorbitol, ethylene glycol, levulinic acid, DL-malic acid, urea, N,N'-dimethylurea, acetamide, glycerol, citric acid, oxalic acid, methylurea, or malonic acid. The hydrogen bond acceptor compounds include one or more of choline chloride, betaine, proline, levulinic acid, glycerol, citric acid, or ethylene glycol. This invention synthesizes various eutectic solvents and systematically investigates their extraction and transformation effects on the effective components of Polygonum multiflorum. These extraction solvents are green, biodegradable, and have great application prospects. They can effectively reduce hepatotoxic components in Polygonum multiflorum and improve the extraction efficiency of its effective components.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for extracting effective components of Radix Polygoni Multiflori based on a deep eutectic solvent system, belonging to the field of traditional Chinese medicine research. BACKGROUND

[0002] Deep eutectic solvents (DESs) as a new generation of green solvents have attracted extensive attention due to their excellent physical and chemical properties. They are not flammable, have good thermal stability, low toxicity, simple preparation, and low raw material prices, which have shown great potential in many fields. Compared with water and traditional organic solvents, DESs have stronger solubility for hydrophilic and hydrophobic compounds, and also exhibit good biocompatibility. DESs are low eutectic mixtures composed of a certain stoichiometric ratio of hydrogen bond donors (HBD) (such as betaine, choline chloride, proline, etc.) and hydrogen bond acceptors (HBA) (such as glycerol, ethylene glycol, etc.). At present, the research on DESs with choline chloride, betaine and proline as hydrogen bond acceptors, and polyols and polyacids as hydrogen bond donors is relatively mature and widely applied. However, the extraction rate of effective components of these methods is not high, and toxic components in the sample are inevitably extracted.

[0003] The Chinese patent application with application number 202210206139.6 and the invention name of a method for selectively extracting stilbene glycosides in Radix Polygoni Multiflori clearly extracts stilbene glycosides, but does not distinguish the hepatotoxic components in Radix Polygoni Multiflori. Therefore, there is an urgent need for a method that can effectively distinguish and extract effective components and hepatotoxic components in Radix Polygoni Multiflori. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a method for extracting effective components of Radix Polygoni Multiflori using a deep eutectic solvent system, which can significantly promote the dissolution of effective components in Radix Polygoni Multiflori and reduce the dissolution of hepatotoxic components in Radix Polygoni Multiflori.

[0005] Technical Solution: To solve the above-mentioned technical problems, this invention provides a method for extracting the effective components of Polygonum multiflorum based on a eutectic solvent system, comprising the following steps: dissolving Polygonum multiflorum powder in a eutectic solvent and extracting it by ultrasonication to obtain the effective components of Polygonum multiflorum; the eutectic solvent includes a hydrogen bond donor compound and a hydrogen bond acceptor compound, wherein the hydrogen bond donor compound includes one or more of xylitol, sorbitol, ethylene glycol, levulinic acid, DL-malic acid, urea, N,N'-dimethylurea, acetamide, glycerol, citric acid, oxalic acid, methylurea, or malonic acid; the hydrogen bond acceptor compound includes one or more of choline chloride, betaine, proline, levulinic acid, glycerol, citric acid, or ethylene glycol; the molar ratio of the hydrogen bond acceptor compound to the hydrogen bond donor compound is 1-5:1-5.

[0006] The molar ratio of the hydrogen bond acceptor compound to the hydrogen bond donor compound is 1-2:1-5.

[0007] The hydrogen bond acceptor compound includes choline chloride or betaine; the hydrogen bond donor compound includes one of sorbitol, xylitol, N,N'-dimethylurea, ethylene glycol, DL-malic acid, urea, or ethylene glycol; and the molar ratio of the hydrogen bond acceptor compound to the hydrogen bond donor compound is 1-5:1-2.

[0008] The hydrogen bond acceptor compound is betaine, the hydrogen bond donor compound is sorbitol, and the molar ratio of betaine to sorbitol is 1:1.2.

[0009] The water content of the eutectic solvent is 10% to 80%. Preferably, the water content is 10% to 70%.

[0010] The liquid-to-solid ratio of the eutectic solvent to the Polygonum multiflorum powder is 5–60:1. Preferably, the liquid-to-solid ratio of the eutectic solvent to the Polygonum multiflorum powder is 30–60:1.

[0011] The ultrasonic extraction time is 1–60 min. Preferably, the ultrasonic extraction time is 10–60 min.

[0012] The power of the ultrasonic extraction is 250-500W.

[0013] The temperature of the ultrasonic extraction is 25–65°C.

[0014] This invention first established quantitative methods for the active ingredients (TTSG, ED, and EME) and hepatotoxic components (CTSG and REG) in Polygonum multiflorum, and conducted methodological investigations, including linearity, intra-day and inter-day precision, repeatability, stability, and recovery rate. The RSD values ​​were all less than 5%, demonstrating that this method can be used for the quantitative analysis of these five components in Polygonum multiflorum. This invention selected seven hydrogen bond acceptors and thirteen hydrogen bond donors, combining them in pairs to successfully synthesize 71 DESs systems with different chemical properties. These systems were used to extract the active ingredients and reduce the hepatotoxic components in Polygonum multiflorum. Key process parameters were optimized, resulting in the optimal process for extracting the active ingredients from Polygonum multiflorum using a Bet / Sor (1:1.2) DESs system. Under optimal extraction conditions, the total extraction rates of the active ingredients and hepatotoxic components were 2.56% and 0.042%, respectively, which are higher than those obtained with conventional solvents (methanol, acetonitrile, ethanol, isopropanol, and water). The Bet / Sor (1:1.2) DESs solvent system helps to promote the extraction of active ingredients from Polygonum multiflorum and reduce the solubility of hepatotoxic components, which can reduce the clinical side effects of Polygonum multiflorum.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention synthesizes a variety of eutectic solvents and systematically investigates the extraction and transformation of effective components in Polygonum multiflorum by a variety of eutectic solvents. The extraction solvents are green and degradable, with great application prospects. They can effectively reduce hepatotoxic components in Polygonum multiflorum and improve the extraction efficiency of effective components in Polygonum multiflorum. Attached Figure Description

[0016] Figure 1 Why are the ultra-high performance liquid chromatograms of Polygonum multiflorum extract and reference standard as follows: Figure 1 A is the ultra-high performance liquid chromatogram of the reference standard; Figure 1 B shows the ultra-high performance liquid chromatogram of Polygonum multiflorum extract; (1: CTSG; 2: TTSG; 3: REG; 4: ED; 5: EME)

[0017] Figure 2 Extraction rates of the target compound for different DESs systems: Figure 2 A and 2C represent the total extraction rates of the active ingredients by different DESs systems; Figure 2 B and 2D represent the extraction rates of hepatotoxic components by different DESs systems;

[0018] Figure 3 Effects of different conditions on the total extraction rate of active ingredients (TTSG, ED, and EME) from Polygonum multiflorum: Figure 3 A represents the effect of solvent water content on the total extraction rate of active ingredients (TTSG, ED, and EME) from Polygonum multiflorum. Figure 3 B represents the effect of the liquid-to-solid ratio on the total extraction rate of effective components (TTSG, ED, and EME) from Polygonum multiflorum.Figure 3 C represents the effect of extraction time on the total extraction rate of effective components (TTSG, ED, and EME) of Polygonum multiflorum; Figure 3 D represents the effect of extraction temperature on the total extraction rate of active ingredients (TTSG, ED, and EME) from Polygonum multiflorum. Figure 3 E represents the effect of ultrasonic frequency on the total extraction rate of effective components (TTSG, ED, and EME) of Polygonum multiflorum.

[0019] Figure 4 Response surface analysis results diagram: Figure 4 A represents the interaction between water content and liquid-to-solid ratio on the extraction rate; Figure 4 B represents the interaction between moisture content and extraction time on the extraction rate;

[0020] Figure 5 The interaction between liquid-to-solid ratio and extraction time on extraction rate;

[0021] Figure 6 Comparison of extraction efficiencies of DESs and different extraction solvents for the effective components (TTSG, ED, and EME) and hepatotoxic components (CTSG and REG) in Polygonum multiflorum: Figure 6 A represents the extraction rate of the active ingredient; Figure 6 B represents the extraction rate of hepatotoxic components. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0023] 1. Experimental apparatus

[0024] The 0.0001 g electronic analytical balance was purchased from Sartorius Group (Beijing, China); the 0.0001 g electronic balance and electronic analytical balance were purchased from Tianma Hengji Instrument Co., Ltd. (Tianjin, China); the KH-500DB digital display ultrasonic cleaner was purchased from Kunshan Hechuang Ultrasonic Instrument Co., Ltd. (Suzhou, China); the Waters ACQUITY ultra-high performance liquid chromatograph and Waters ACQUITY UHPLC-AB 4600QTOF MS; the Eppendorf 5425R high-speed refrigerated centrifuge and pipettes were purchased from Eppendorf (Hamburg, Germany); the 0.45 μm organic filter was purchased from Tianjin Linghang Experimental Equipment Co., Ltd. (Tianjin, China); and the HJ-4A multi-head magnetic heating stirrer was purchased from Guohua Electric Co., Ltd. (Changzhou, China).

[0025] 2. Experimental reagents and chemicals

[0026] The Polygonum multiflorum root was purchased from the Bozhou Medicinal Materials Market in Anhui Province and identified as the dried tuberous root of Polygonum multiflorum Thunb., a plant of the Polygonaceae family. The Polygonum multiflorum root was pulverized and passed through a 50-mesh sieve. The resulting powder was placed in a brown glass bottle and stored in a refrigerator at 4°C.

[0027] Chromatographically pure methanol was purchased from Merck (Darmstadt, Germany); chromatographically pure formic acid was purchased from ROE (Newark, USA); analytically pure methanol and anhydrous ethanol were purchased from Nanjing Chemical Reagent Co., Ltd. (Nanjing, China); deionized water (18 MΩ·cm) was purified using a Milli-Q water purification system (Millipore, Massachusetts, USA).

[0028] Reference standards: cis-stilbene glycoside (CTSG, purity >98%), trans-stilbene glycoside (TTSG, purity >98%), emodin-8-glucoside (REG, purity >98%), emodin (ED, purity >98%), and emodin methyl ether (EME, purity >98%) were all purchased from Chengdu Pusi Biotechnology Co., Ltd. (Chengdu, China). The structural formulas of each reference standard are shown below, and the batch numbers of each reference standard are shown in Table 1.

[0029]

[0030] Table 1. Batch numbers of different reference standards

[0031]

[0032] Analytical grade reagents: Choline chloride (ChCl), anhydrous betaine (Bet), L-proline (Pro), DL-malic acid (Ma), urea (Ure), levulinic acid (La), xylitol (Xyl), D-sorbitol (Sor), N,N'-dimethylurea (Dim), acetamide (Ace), citric acid (Ca), oxalic acid (Oa), methylurea (Met), malonic acid (Pa), and glycerol (Gly) were all purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Ethylene glycol (Eth) was purchased from Nanjing Chemical Reagent Co., Ltd. (Nanjing, China). The purity of all reagents was above 98%. The batch numbers of each reagent are shown in Table 2.

[0033] Table 2 Batch numbers of different reagents

[0034]

[0035] Example 1 Methodological Investigation

[0036] 1. Sample solution preparation

[0037] Accurately weigh 1g of Polygonum multiflorum powder and place it in an Erlenmeyer flask. Add 30mL of methanol and extract by ultrasonication at room temperature (40KHz, 500W) for 60min. After centrifuging the extract at 13500rcf for 10min, filter it through a 0.22μm filter membrane and collect the supernatant in a vial for sample injection analysis.

[0038] 2. Preparation of reference solution

[0039] Five compound reference standards were accurately weighed and dissolved in methanol to prepare solutions containing 37.50 μg CTSG, 1875.00 μg TTSG, 118.75 μg REG, 76.88 μg ED, and EME per mL. A 47.19 μg mixed reference standard stock solution was serially diluted with methanol to prepare working solutions of the reference standards (CTSG concentrations were 1.17 μg / mL, 2.34 μg / mL, 4.69 μg / mL, 9.38 μg / mL, 18.75 μg / mL, and 37.50 μg / mL; TTSG concentrations were 58.59 μg / mL, 117.19 μg / mL, 234.38 μg / mL, 468.75 μg / mL, 937.50 μg / mL, and 1875.00 μg / mL; REG concentrations were 1.86 μg / mL, 3.71 μg / mL, and 18.75 μg / mL). The concentrations of analytes were 7.42 μg / mL, 14.85 μg / mL, 29.69 μg / mL, and 59.38 μg / mL; the concentrations of ED were 1.20 μg / mL, 2.40 μg / mL, 4.81 μg / mL, 9.61 μg / mL, 19.22 μg / mL, and 38.44 μg / mL; and the concentrations of EME were 1.47 μg / mL, 2.95 μg / mL, 5.90 μg / mL, 11.80 μg / mL, and 23.59 μg / mL. These compounds were analyzed by liquid chromatography, and linear regression analysis was performed using the peak area of ​​the analytes against the concentrations to obtain standard curves for each compound. Based on pharmacopoeia and literature, acetonitrile (A) – 0.1% formic acid aqueous solution (B) was selected as the mobile phase, with a column temperature of 45℃, a flow rate of 0.3 mL / min, an injection volume of 1 μL, and a detection wavelength of 254 nm. The elution gradient was optimized, and the optimal elution program was obtained as follows: 0-1 min, 10% A; 1-5 min, 10-16% A; 5-13 min, 16-38% A; 13-17 min, 38-95% A; 17-20 min, 95% A; post-column equilibration for 3 min. A Waters Acquity UPLC system equipped with a VWD detector, binary pump, and column oven was used. The chromatographic column was a Waters Acquity UPLC BEHC. 18 (2.1×100mm, 1.8μm). The liquid chromatograms of the mixed reference solution (A) and the Polygonum multiflorum extract sample solution (B) are shown below. Figure 1 As shown in Table 3, the linear relationship of the target compounds in Polygonum multiflorum was investigated. Table 3 shows that the target compounds exhibit good linearity within a certain concentration range (R0). 2 ≥0.9991.

[0040] Table 3 Standard curves of analytes in Polygonum multiflorum

[0041]

[0042] Example 2 Precision Examination

[0043] Intra-day precision: The reference working solution from Example 1 was injected 6 times consecutively within one day. The retention time and peak area of ​​the target compound were recorded, and the relative standard deviation (RSD) was calculated.

[0044] Daytime precision: The above reference solution was injected three times a day for three consecutive days. The retention time and peak area of ​​each compound were recorded, and the relative standard deviation (RSD) was calculated.

[0045] The precision results of the analytes are shown in Table 4. The RSD of the intra-day precision of the five analytes ranged from 0.12% to 3.61%, and the RSD of the inter-day precision ranged from 1.06% to 4.05%, both less than 5%, indicating that the intra-day and inter-day precision of this method are good.

[0046] Table 4. Repeatability, stability, and accuracy of the tested components in Polygonum multiflorum.

[0047]

[0048] Example 3 Repeatability Test

[0049] Following the sample preparation method in Example 1, six parallel sample solutions of Polygonum multiflorum were prepared. The six samples were subjected to liquid chromatography analysis under the chromatographic conditions of Example 1. The retention time and peak area of ​​the target analyte were recorded, and the RSD value of each target peak was calculated to examine the repeatability of the samples. The results are shown in Table 4. The results indicate that the RSD values ​​of the target compounds in Polygonum multiflorum were all less than 2.83%, indicating that the method has good repeatability.

[0050] Example 4 Stability Study

[0051] The samples prepared according to the sample preparation method in Example 1 were subjected to liquid chromatography analysis at 0h, 4h, 6h, 8h, 12h and 24h according to the chromatographic conditions of Example 1. The peak areas of each compound were recorded, the RSD values ​​were calculated, and the stability of the samples was examined. The results are shown in Table 4. The results show that the RSD values ​​of the peak areas of the target compounds in Polygonum multiflorum were all less than 4.26%, indicating that the sample solutions were stable within 24h.

[0052] Example 5 Accuracy Examination

[0053] The accuracy was assessed using the spiking recovery method: a precise amount of Polygonum multiflorum powder was weighed in six parallel samples, and a reference standard equivalent to the sample content of the analyte was added to each sample. Samples were prepared according to the sample preparation method in Example 1, and liquid chromatography analysis was performed under the chromatographic conditions of Example 1. The spiking recovery rate and RSD value of each compound were calculated. The formula for calculating the spiking recovery rate is: Recovery rate (%) = (Actual measured amount - Content of reference standard in the medicinal material) / Amount of reference standard added × 100%. The results are shown in Table 4. The average recoveries of the analytes in Polygonum multiflorum ranged from 97.51% to 104.16%, and the RSD values ​​ranged from 1.86% to 4.29%, meeting the pharmacopoeia requirements, indicating that the method has good accuracy.

[0054] Example 6: Screening of the optimal extraction system based on DESs

[0055] 1. Preparation of DESs

[0056] First, 71 DESs were synthesized according to Table 5. Based on the specific molar ratio, appropriate amounts of HBD and HBA were weighed into the same beaker, heated to 70°C, and stirred continuously at this temperature until a homogeneous and clear liquid was formed. After cooling to room temperature, the liquid was placed in a desiccator for storage and later use.

[0057] Table 5 shows the synthesis of 71 types of DESs.

[0058]

[0059]

[0060] 2. Optimal DESs system selection

[0061] First, a series of solvent systems (1-39) based on the aforementioned DESs were prepared, totaling 71 DESs. Each DES-based solvent system consisted of 50% DESs (v / v) and 50% water (v / v). 1 g of Polygonum multiflorum powder was accurately weighed and added to 30 mL of the prepared solvent system. The mixture was extracted by sonication (40 kHz, 500 W, 30 °C) for 30 min. The extract was centrifuged at 13000 rpm for 10 min, filtered through a 0.45 μm microporous membrane, and analyzed by liquid chromatography under the conditions of Example 1. Based on the DESs systems of choline chloride, betaine, proline, levulinic acid, glycerol, citric acid, and ethylene glycol, the optimal DES was selected based on the highest total extraction rates of TTSG, ED, and EME (active components) and the lowest total extraction rates of CTSG and REG (hepatotoxic components) from Polygonum multiflorum.

[0062] like Figure 2As shown in Figures A and 2C, the ordinate represents the extraction yields (mg / g) of three active components (TTSG, ED, and EME) in Polygonum multiflorum under different systems. Compared with methanol extraction, it was found that 28 DESs solvent systems could effectively improve the total extraction yields of TTSG, ED, and EME (from a minimum of 22.12 to a maximum of 26.65 mg / g). Among these DESs systems, the DESs solvent systems with CA as the hydrogen bond acceptor and Sor as the hydrogen bond donor (except for ChCl / Sor (3:1)) could effectively improve the extraction yields of these active components in Polygonum multiflorum. The order of the total extraction yields of these three active components in different DESs systems from high to low is: Ca / Ace (1:2) > Ca / Ace (1:1) > Bet / Sor (1:1.2) > Gly / Ace (1:1) > Ca / Dim (2:1) > ChCl / Sor (2:1) > Eth / Ace (2:1) > Pro / Gly (2:5) > Bet / Ca (2:1) > Ca / La (1:2) (top 10).

[0063] In addition, the total extraction yields of the main hepatotoxic components CTSG and REG in Polygonum multiflorum were investigated for 71 DESs solvent systems, and the results are shown in Figure 2 Figures B and 2D. The ordinate represents the total extraction yields (mg / g) of two hepatotoxic components (CTSG and REG) in Polygonum multiflorum. Compared with methanol extraction, except for Bet / CA (2:1), CA / Ace (1:2), CA / Dim (2:1), and La / Ure (2:1), the other 67 DESs systems could significantly reduce the total extraction yields of CTSG and REG. The order of the total extraction yields of these two hepatotoxic components in different DESs systems from low to high is: ChCl / Sor (3:1) < ChCl / Xyl (5:2) < ChCl / Dim (1:1) < Bet / Sor (1:1.2) < ChCl / Sor (2:1) < ChCl / Eth (2:1) < ChCl / Eth (1:1) < ChCl / Ma (2:1) < ChCl / Ure (1:2) < ChCl / Eth (1:2) (top ten).

[0064] In summary, the DESs system using CA as the hydrogen bond acceptor can effectively improve the total extraction rate of the active ingredients TTSG, ED, and EM from Polygonum multiflorum, but it also promotes the dissolution of hepatotoxic components CTSG and REG. The DESs solvent system using Sor as the hydrogen bond donor (except for ChCl / Sor(3:1)) can significantly reduce the total extraction rate of hepatotoxic components CTSG and REG, while promoting the total dissolution of the active ingredients TTSG, ED, and EM. Among the DESs systems tested using Sor as the hydrogen bond donor, Bet / Sor(1:1.2) showed the highest efficiency in promoting the dissolution of active ingredients and reducing the dissolution of hepatotoxic components from Polygonum multiflorum; therefore, Bet / Sor(1:1.2) was selected for further experiments.

[0065] Example 7: Single Factor Analysis

[0066] Under the conditions of Example 6, Bet / Sor (1:1.2) showed a high extraction rate of effective components and a low extraction efficiency of hepatotoxic components from Polygonum multiflorum. Therefore, Bet / Sor (1:1.2) was selected for subsequent studies. Subsequently, using the total extraction rate of TTSG, ED, and EME from Polygonum multiflorum as an indicator, a single-factor experimental design was employed to optimize key extraction parameters and determine the optimal extraction conditions.

[0067] 1. DESs moisture content investigation

[0068] Accurately weigh an appropriate amount of Polygonum multiflorum powder and extract it using DES:water mixed solutions of different concentrations (10%, 20%, 30%, 40%, 50%, 60%, 70%, and 80%) as extraction solvents at a liquid-to-solid ratio of 20:1 mL / g. Extract by ultrasonication (40 kHz, 500 W, 35℃) for 30 min. After extraction, centrifuge the extract at 13500 rcf for 10 min, collect the supernatant, filter it through a 0.22 μm filter membrane, and collect the filtrate for analysis according to the conditions in Example 1. Eutectic groups generally have high viscosity, requiring the addition of a certain amount of water to reduce the viscosity of the extraction solvent system, thereby facilitating the extraction of active ingredients. Figure 3 As shown in Figure A, the extraction rate of active ingredients in Polygonum multiflorum gradually increased when the water content of DESs increased from 20% to 50%; however, the extraction rate gradually decreased when the water content of DESs increased from 50% to 80%. Based on the total extraction rate of active ingredients in Polygonum multiflorum, a solvent system with 50% DESs was selected.

[0069] 2. Liquid-to-solid ratio investigation

[0070] Accurately weigh an appropriate amount of Polygonum multiflorum powder and extract it using a 50% Bet / Sor (1:1.2):water mixture as the extraction solvent. The liquid-to-solid ratios were 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, and 60:1 mL / g. Extraction was performed ultrasonically (40 kHz, 500 W, 30 °C) for 30 min. After extraction, the extract was centrifuged at 13500 rcf for 10 min. The supernatant was collected, filtered through a 0.22 μm filter membrane, and the filtrate was analyzed under the conditions described in Example 1. Figure 3 As shown in Figure B, the extraction rate of the target compound gradually increased as the liquid-to-solid ratio increased from 5:1 mL / g to 40:1 mL / g. However, when the liquid-to-solid ratio continued to increase from 40:1 mL / g to 60:1 mL / g, the extraction rate of the target compound decreased. Therefore, 40:1 mL / g was selected as the optimal liquid-to-solid ratio.

[0071] 3. Examination of extraction time

[0072] Accurately weigh an appropriate amount of Polygonum multiflorum powder and extract it using a Bet / Sor (1:1.2):water mixture (50%) as the extraction solvent at a liquid-to-solid ratio of 40:1 mL / g. Extract the powder by ultrasonication (40 kHz, 500 W, 35 °C) for 1 min, 5 min, 10 min, 20 min, 30 min, 40 min, and 60 min, respectively. After extraction, centrifuge the extract at 13500 rcf for 10 min, collect the supernatant, filter it through a 0.22 μm filter membrane, and analyze the filtrate according to the conditions described in Example 1. Figure 3 As shown in Figure C, the extraction rate of the target compound increased when the extraction time increased from 1 min to 20 min, and decreased when the extraction time increased from 20 min to 60 min. Therefore, the optimal extraction time was selected as 20 min.

[0073] 4. Extraction temperature investigation

[0074] Accurately weigh an appropriate amount of Polygonum multiflorum powder and extract it using a Bet / Sor (1:1.2):water mixture (50%) as the extraction solvent at a liquid-to-solid ratio of 40:1 mL / g. Extract by ultrasonication (40 kHz, 500 W) for 20 min at extraction temperatures of 25℃, 35℃, 45℃, 55℃, and 65℃. After extraction, centrifuge the extract at 13500 rcf for 10 min, collect the supernatant, filter it through a 0.22 μm filter membrane, and analyze the filtrate according to the conditions described in Example 1. Figure 3 As shown in Figure E, the extraction rate of the target compound increased when the extraction temperature was increased from 25℃ to 35℃. However, when the temperature continued to rise to 65℃, the extraction rate of the target compound decreased. This may be because stilbene glycosides are glycosides, which have poor stability at higher temperatures. Therefore, 35℃ is the optimal choice to ensure extraction efficiency.

[0075] 5. Ultrasonic power assessment

[0076] Accurately weigh an appropriate amount of Polygonum multiflorum powder and extract it using a Bet / Sor (1:1.2):water mixture (50%) as the extraction solvent. The liquid-to-solid ratio is 40:1 mL / g. Extract by ultrasonication (40 kHz, 30℃) for 20 min at ultrasonic powers of 250 W, 300 W, 350 W, 400 W, 450 W, and 500 W. After extraction, centrifuge the extract at 13500 rcf for 10 min, collect the supernatant, filter it through a 0.22 μm filter membrane, and analyze the filtrate according to the conditions in Example 1. Figure 3 As shown in D, the ultrasonic power has little effect on the extraction rate of the target compound. When the ultrasonic power increases from 250W to 450W, the extraction rate of the target compound increases; when the ultrasonic power increases from 450W to 500W, the extraction rate of the target compound does not change significantly. Therefore, the optimal ultrasonic power is selected as 450W.

[0077] Example 8 Response Surface Analysis

[0078] Based on the results of single-factor experiments, and using the Box-Behnken experimental design principle, three factors at three levels were selected for optimization. The three factors were denoted as A (water content in the extraction solvent, %), B (material ratio, mg / mL), and C (extraction time). The high, medium, and low levels of each factor were denoted as 1, 0, and -1, respectively. The levels of each factor are shown in Table 6. Using the total content of TTSG, ED, and EME in *Polygonum multiflorum* as the response value, response surface analysis was performed using a Box-Behnken experimental design established with Design Expert software.

[0079] Table 6. Experimental Design of Box-Behnken

[0080]

[0081] The extraction rates of TTSG, ED and EM measured under 17 different conditions are shown in Table 7.

[0082] Table 7 Experimental Design and Results of Response Surface

[0083]

[0084]

[0085] The experimental results in the table were subjected to multiple linear regression and binomial fitting. The mathematical simulation of the total extraction rates of TTSG, ED, and EM in Polygonum multiflorum is as follows: Y = 2.574 + 0.119A + 0.079B - 0.014C - 0.091AB - 0.009BC + 0.062AC - 0.133A 2 -0.090B 2 -0.078C 2

[0086] In the formula, Y represents the extraction efficiency (%), and A, B, and C represent the DESs water content, liquid-solid ratio, and extraction time, respectively, as set by the extraction conditions.

[0087] Analysis of variance was performed on the above regression equations, and the results are shown in Table 8. When P < 0.05, the influence of factors on the response values ​​was significant; when P < 0.001, the difference was highly significant. The model in this experiment was significant (P < 0.001), indicating that the regression model indicators were highly significant. The lack of fit term was used to assess the model fit; if it was not significant, it indicated that the equation simulation was good. The results showed that the lack of fit term P = 0.305 > 0.05, indicating a good model fit; the correlation coefficient R... 2 =0.928, further demonstrating that the model has good reliability.

[0088] Table 8. Analysis of Variance Table of Regression Equations

[0089]

[0090] The analysis results of the extraction conditions of Polygonum multiflorum are as follows: Figure 4 and Figure 5 As shown, the water content and liquid-to-solid ratio of the DES significantly affected the extraction of active ingredients (P<0.01). The extraction efficiency of active ingredients (TTSG, ED, and EM) in Polygonum multiflorum increased continuously until the water content in the extraction solvent reached 45%; however, when the water content in the extraction solvent exceeded 45%, the extraction efficiency of active ingredients showed a negative correlation. The extraction efficiency of active ingredients increased with the increase of the material ratio to 40%; however, when the material ratio exceeded 40%, the change in extraction efficiency of active ingredients was not significant. The extraction time (variable C) had a relatively small effect on the extraction efficiency (P=0.560).

[0091] The extraction method was optimized using Design Expert software. The optimized extraction conditions were: water content in DES of 46.22%, material ratio of 42.50 mg / mL, and extraction time of 20.76 min. Under these optimized conditions, the predicted total extraction efficiency of active ingredients (TTSG, ED, and EM) from Polygonum multiflorum was 2.61%. Experiments were conducted to verify the extraction of active ingredients (TTSG, ED, and EM) from Polygonum multiflorum under the above optimized conditions. The average extraction efficiency of three experiments was 2.56%, which is close to the theoretical value, indicating that the model is reliable.

[0092] Example 8: Comparative Analysis of Extraction with Different Solvents

[0093] An appropriate amount of Polygonum multiflorum powder was accurately weighed and extracted using methanol, acetonitrile, ethanol, isopropanol, and water as extraction solvents at a liquid-to-solid ratio of 40:1 mL / g. The mixture was ultrasonically extracted (40 kHz, 450 W, 35 °C) for 20 min. After extraction, the extract was centrifuged at 13500 rcf for 10 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane. The filtrate was then analyzed under the conditions described in Example 1. The results are as follows: Figure 6 As shown, the total extraction rate of the active ingredients is in the following order: DESs > methanol > water > ethanol > acetonitrile > isopropanol. Figure 6 A); The order of total extraction rates of hepatotoxic components was: methanol > ethanol > water > acetonitrile > DESs > isopropanol ( Figure 6 B). In summary, compared with conventional extraction solvents, the optimized DESs (Bet / Sor) showed the highest extraction efficiency for the active ingredients in Polygonum multiflorum, but a lower extraction efficiency for hepatotoxic components.

Claims

1. A method for extracting effective components of Radix Polygoni Multiflori based on a eutectic solvent system, characterized in that, The method comprises the following steps: The effective components of the polygonum multiflorum thunb are obtained by dissolving the polygonum multiflorum thunb powder with a eutectic solvent, and ultrasonic extraction, wherein the eutectic solvent comprises a hydrogen bond donor compound and a hydrogen bond acceptor compound, the hydrogen bond acceptor compound is betaine, the hydrogen bond donor compound is sorbitol, and the molar ratio of the betaine to the sorbitol is 1:1.

2.

2. The method of claim 1, wherein, The water content of the eutectic solvent is 10%-80%.

3. The method of claim 1, wherein, The liquid-solid ratio of the eutectic solvent to the polygonum multiflorum thunb powder is 5-60:

1.

4. The method of claim 1, wherein, The liquid-solid ratio of the eutectic solvent to the polygonum multiflorum thunb powder is 30-60:

1.

5. The method of claim 1, wherein, The power of the ultrasonic extraction is 250-500 W.

6. The method of claim 1, wherein, The temperature of the ultrasonic extraction is 25-65 DEG C.

7. The method of claim 1, wherein, The time of the ultrasonic extraction is 1-60 min.

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