Ophiopogon japonicus polysaccharide extraction method

Abstract

The invention discloses an ophiopogon japonicus polysaccharide extraction method, which comprises: (1) carrying out extraction on ophiopogon japonicus and distilled water according to a solid-liquid ratio of 1: 40-70 in an electromagnetic heating extractor under a normal pressure at a temperature of 100 DEG C for 60-80 min each time, removing drug residue and silt after each extraction, and combining the extraction solutions; (2) concentrating the mixed extracting solution by using a rotary evaporator until the dry weight volume ratio of the radix ophiopogonis is 2: 1; (3) adding equal volume of dichloromethane into the concentrated solution for primary extraction; (4) carrying out secondary extraction on the primary extract and petroleum ether according to a volume ratio of 2.5: 1-1: 1; (5) adding a proper amount of distilled water into the secondary extracting solution, and concentrating again until the dry weight volume ratio of the radix ophiopogonis is 2: 1; (6) adding 95% ethanol into the secondary concentrated solution according to an alcohol precipitation ratio of 1: 3-1: 4.5, and carrying out alcohol precipitation in a refrigerator at 5 DEG C overnight; and (7) after the alcohol precipitation is completed, discarding the supernate, collecting the precipitate, and drying until no alcohol smell exists. According to the method, the polysaccharide extraction rate is greatly increased.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine extraction technology, specifically to a method for extracting polysaccharides from Ophiopogon japonicus. Background Technology

[0002] As one of the four basic substances that make up the human body, polysaccharides have been studied relatively late compared to the three basic substances of proteins, nucleic acids, and lipids.

[0003] Currently, common methods for extracting polysaccharides from Ophiopogon japonicus include solvent extraction, ultrasonic extraction, pulsed electric field extraction, and high-pressure high-temperature extraction. Each method has its advantages: solvent extraction is simple to operate, ultrasonic-assisted extraction accelerates dissolution, pulsed electric field extraction efficiently breaks down cell walls, and high-pressure high-temperature extraction promotes polysaccharide release through high temperature and pressure. However, despite the widespread application of these technologies in production, there is still room for improvement in extraction rates. Some methods suffer from unstable extraction efficiency due to limitations in process parameters or high equipment costs. Therefore, it is urgent to further optimize process parameters and explore new extraction technologies to improve the extraction efficiency of Ophiopogon japonicus polysaccharides, addressing the shortcomings of existing extraction techniques. Summary of the Invention

[0004] The present invention aims to provide a method for extracting Ophiopogon japonicus polysaccharides, so as to improve the extraction efficiency of Ophiopogon japonicus polysaccharides.

[0005] This invention provides a method for extracting polysaccharides from Ophiopogon japonicus, comprising the following steps: (1) Mix Ophiopogon japonicus with distilled water at a material-liquid ratio of 1:40-70 and extract twice under normal pressure in an electromagnetic heating extractor. The extraction temperature is 100℃ and the extraction time is 60-80min each time. After each extraction, filter twice with medical gauze while hot, and then filter twice with fine filter paper. Combine the extracts to obtain a mixed extract. (2) The mixed extract was concentrated using a rotary evaporator until the dry weight volume ratio of Ophiopogon japonicus was 2:1 to obtain a first-concentrated solution; (3) Add an equal volume of dichloromethane to the primary concentrate, shake thoroughly to extract, let stand and separate the layers, discard the lower dichloromethane phase, retain the upper aqueous phase, and obtain the decolorized primary extract. (4) The primary extract was subjected to a secondary extraction with petroleum ether at a volume ratio of 2.5:1 to 1:1. After standing and separating the layers, the upper petroleum ether phase was discarded and the lower aqueous phase was retained to obtain the secondary extract. (5) Add an appropriate amount of distilled water to the secondary extract and concentrate it again until the dry weight volume ratio of Ophiopogon japonicus is 2:1 to obtain the secondary concentrate. (6) Add 95% ethanol to the secondary concentrate at an alcohol precipitation ratio of 1:3-1:4.5 while stirring, and then place it in a refrigerator at 5°C for alcohol precipitation overnight; (7) After the alcohol precipitation is completed, discard the supernatant, collect the precipitate and dry it until there is no alcohol smell.

[0006] Furthermore, in step (1), the material-to-liquid ratio is 1:55.

[0007] Furthermore, the extraction time in step (1) is 75 minutes each time.

[0008] Furthermore, the volume ratio in step (4) is 2:1.

[0009] Furthermore, in step (6), the alcohol precipitation ratio is 1:4.

[0010] Furthermore, the electromagnetic heating extractor is model YJDJ08.

[0011] The beneficial effects of this invention are: This invention employs electromagnetic heating extraction technology, using the extraction rate of Ophiopogon japonicus polysaccharides as the evaluation index. A single-factor experimental system was used to systematically investigate the effects of material-to-liquid ratio, extraction time, petroleum ether volume ratio, and alcohol precipitation ratio on the extraction efficiency. Based on L9(3 4 Orthogonal experiments optimized the process parameters and conducted systematic methodological verification, which ultimately significantly improved the problems of low efficiency, high energy consumption and insufficient polysaccharide conversion rate in the traditional Ophiopogon japonicus polysaccharide extraction process. It achieved a significant increase in polysaccharide extraction rate and provided an efficient, stable and replicable process solution for the development of traditional Chinese medicine polysaccharide preparations, which has important application value and promotion prospects. Attached Figure Description

[0012] Figure 1 This is a scan spectrum of glucose at its maximum wavelength; Figure 2 For glucose standard curve; Figure 3 Figure 1 shows the effect of different material-to-liquid ratios on the extraction rate of Ophiopogon japonicus polysaccharides. Figure 4 Figure showing the effect of different extraction times on the extraction rate of Ophiopogon japonicus polysaccharides; Figure 5 Figure 1 shows the effect of different volume ratios on the extraction rate of Ophiopogon japonicus polysaccharides. Figure 6 Figure 1 shows the effect of different alcohol precipitation ratios on the extraction rate of Ophiopogon japonicus polysaccharides. Figure 7 This is a graph showing the average values ​​of each factor. Detailed Implementation

[0013] The following detailed description, with reference to specific embodiments, further illustrates the above-described content of the present invention. However, the scope of protection of the present invention is not limited to the embodiments described herein.

[0014] 1. The instruments and reagents involved in this invention are as follows: 1.1 Instruments: YJDJ08 electromagnetic heating extractor (Changsha Zhuocheng Medical Instrument Co., Ltd.), RE-52 rotary evaporator (Shanghai Yarong Biochemical Instrument Factory), SQP analytical balance (Sartorius Scientific Instruments Co., Ltd., sensitivity 0.01mg), HH-2 digital display constant temperature water bath (Changzhou Langyue Instrument Manufacturing Co., Ltd.), JP-040S ultrasonic cleaner (Shenzhen Jiemeng Cleaning Equipment Co., Ltd.), ZNHW electric heating mantle (Gongyi Yuhua Instrument Co., Ltd.), SHB-3 circulating water multi-purpose vacuum pump (Zhengzhou Dufu Instrument Factory), UV-1800 ultraviolet spectrophotometer (Shanghai Meipuda Instrument Co., Ltd.), GZX-9023MB capacitor forced-air drying oven (Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory).

[0015] 1.2 Reagents: Glucose (analytical grade, Xilong Scientific Co., Ltd., HY250429), concentrated sulfuric acid (analytical grade, Nanchang Xinguang Fine Chemical Reagent Co., Ltd., G286250526), ​​anthrone (analytical grade, Shanghai Maclean Biochemical Technology Co., Ltd., C11882592), dichloromethane (analytical grade, Xilong Scientific Co., Ltd., SJ250323), petroleum ether (analytical grade, Xilong Scientific Co., Ltd., SJ250716), 95% ethanol (analytical grade, Xilong Scientific Co., Ltd., SJ250521).

[0016] 1.3 Medicinal Material: Sichuan Ophiopogon japonicus, produced by Anhui Tongtai Tuozutang Pharmaceutical Co., Ltd., production batch number: YP25020118, the quality standard of the processed slices is "Beijing Municipal Standard for Processing of Chinese Herbal Medicine Slices", and it was identified as processed slices of Sichuan Ophiopogon japonicus by Professor Zhang Jinlian of the Department of Processing of Chinese Herbal Medicine, School of Pharmacy, Jiangxi University of Traditional Chinese Medicine.

[0017] 2. Method for determining the extraction rate of Ophiopogon japonicus polysaccharides 2.1 Construction of the glucose standard curve (1) Experimental principle Glucose rapidly dehydrates to form furfural under the action of concentrated sulfuric acid. Furfural can then dehydrate and condense with anthrone to form a blue-green furfural derivative. The color intensity of this derivative solution at the maximum absorption wavelength in the visible light region is directly proportional to the mass concentration of glucose. The reaction equation is as follows: .

[0018] (2) Determination of the maximum absorption wavelength: The sample solution after the sulfuric acid-anthrone reaction was scanned in the wavelength range of 400-800 nm using a UV-Vis spectrophotometer to determine the maximum absorption wavelength of glucose. The maximum absorption wavelength of glucose was scanned as follows: Figure 1As shown in the figure, there are three absorption peaks at 450 nm, 490 nm, and 628 nm. Among these three absorption peaks, the maximum absorption wavelength is 628 nm, and interference from end absorption can be avoided at this wavelength. Therefore, 628 nm is the most suitable wavelength for measurement.

[0019] Preparation of 80% H2SO4-anthrone: Weigh 0.20g of anthrone using an analytical balance, dissolve it in 25ml of 80% H2SO4, store in a brown bottle protected from light, and prepare fresh each time.

[0020] Sample solution preparation: Take a stoppered tube and add 1 mL of 0.1 mg / mL glucose solution and 4 mL of 80% H2SO4-anthrone. After adding the solution, immediately stop the reaction in a cold water bath. Finally, place the stoppered tube in a 100℃ water bath for 20 min. After the reaction is complete, immediately stop the reaction in a cold water bath to obtain the sample solution for scanning the maximum absorption wavelength of glucose.

[0021] Preparation of the reference solution: Take another stoppered tube, add 1.0 mL of distilled water and 4 mL of 80% H2SO4-anthrone, and perform the remaining operations in the same way as the sample solution.

[0022] (3) Drawing up the glucose standard curve Preparation of standard glucose solution: Weigh 0.10 g of glucose standard using an analytical balance, dissolve it in a 100 mL volumetric flask, and dilute to volume to prepare a 1 mg / mL stock solution; then use a pipette to transfer 4, 6, 8, 10, 12, 14, and 16 mL of the stock solution into 100 mL volumetric flasks and dilute to volume again to prepare glucose solutions of 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, and 0.16 mg / mL, respectively.

[0023] Method for constructing the standard curve: Take 8 stoppered tubes and number them 0, 1, 2, 3, 4, 5, 6, and 7. Add 1 mL of distilled water and the corresponding standard glucose solution of different concentrations to each tube, and then perform the sulfuric acid-anthrone reaction. Finally, measure the absorbance of each glucose concentration at the maximum absorption wavelength, and perform linear regression of the corresponding absorbance value against the glucose concentration to obtain the glucose standard curve equation.

[0024] The absorbance A of each glucose mass concentration was measured at a wavelength of 628 nm, and the regression equation for the glucose standard curve was obtained: y = 4.8578x + 0.0031. Figure 2 As shown.

[0025] 2.2 Determination of Ophiopogon japonicus polysaccharide extraction rate (1) Measurement principle Polysaccharides are first hydrolyzed into monosaccharides under the action of concentrated sulfuric acid, and the color development principle of monosaccharides is the same as described above. The concentration of polysaccharides in the test sample is calculated based on the regression equation of the glucose standard curve, and then the extraction rate of polysaccharides is calculated.

[0026] (2) Preparation of polysaccharide test samples and determination of their absorbance Preparation of polysaccharide test sample: Take dried polysaccharide, dissolve it in an appropriate amount of distilled water, transfer it to a 1000ml volumetric flask and make up to volume to obtain polysaccharide stock solution; then take 1mL from the polysaccharide stock solution and make up to volume in a 100mL volumetric flask to obtain polysaccharide test sample.

[0027] Absorbance measurement: Take two stoppered tubes, labeled 0 and 8. Add 1.0 mL of distilled water to tube 0 and 1.0 mL of polysaccharide test solution to tube 8. Then add 4.0 mL of 80% distilled water to each tube. - Anthrone solution, mix well immediately and react in a 100℃ water bath for 20 min. After the reaction is complete, immediately cool to room temperature in an ice bath. Use tube 0 as a blank control and measure the absorbance value of tube 8 at the maximum absorption wavelength. Example 1

[0028] This embodiment provides a method for extracting polysaccharides from Ophiopogon japonicus, including the following steps: (1) Weigh 25g of dried Ophiopogon japonicus, add 55 times the amount of distilled water, and extract twice in an electromagnetic heating extractor. The extraction temperature is 100℃ under normal pressure, and the extraction time is 75min each time. After each extraction, filter twice with medical gauze while hot, and then filter twice with fine filter paper to remove the dregs and mud. Combine the extracts to obtain a mixed extract. (2) The mixed extract was concentrated to 12.5 ml using a rotary evaporator to obtain a first concentrate; (3) Add an equal volume of dichloromethane to the first concentrate, shake thoroughly to extract, let stand and separate the layers, discard the lower dichloromethane phase, retain the upper aqueous phase, and obtain the decolorized first extract. (4) The primary extract was extracted with petroleum ether at a volume ratio of 2:1. After standing and separating the layers, the upper petroleum ether phase was discarded and the lower aqueous phase was retained to obtain the secondary extract in order to remove fat-soluble impurities. (5) Add distilled water to the secondary extract until the total volume is 25 ml, and concentrate it again to 12.5 ml to obtain a secondary concentrate to remove residual extractant; (6) Add 95% ethanol to the secondary concentrate at an alcohol precipitation ratio of 1:4 while stirring, and then place it in a refrigerator at 5°C for alcohol precipitation overnight. (7) After the alcohol precipitation is completed, discard the supernatant, collect the precipitate and dry it until there is no alcohol smell. Example 2

[0029] Single-factor experiment on the effect of material-liquid ratio on extraction rate This experiment aimed to investigate the effect of different material-to-liquid ratios on the extraction rate of Ophiopogon japonicus polysaccharides. The experimental steps are as follows: (1) Weigh 25g of dried Ophiopogon japonicus and add distilled water at material-liquid ratios of 1:30, 1:40, 1:50, 1:60 and 1:70 respectively. Extract twice in an electromagnetic heating extractor at 100℃ under normal pressure for 60 minutes. After each extraction, filter twice with medical gauze while hot, and then filter twice with fine filter paper to remove the dregs and mud. Combine the extracts to obtain a mixed extract. (2) The mixed extract was concentrated to 12.5 ml using a rotary evaporator to obtain a first concentrate; (3) Add an equal volume of dichloromethane to the first concentrate, shake thoroughly to extract, let stand and separate the layers, discard the lower dichloromethane phase, retain the upper aqueous phase, and obtain the decolorized first extract. (4) The first extract was extracted with petroleum ether at a volume ratio of 2:3. After standing and separating the layers, the upper petroleum ether phase was discarded and the lower aqueous phase was retained to obtain the second extract in order to remove fat-soluble impurities. (5) Add distilled water to the secondary extract until the total volume is 25 ml, and concentrate it again to 12.5 ml to obtain a secondary concentrate to remove residual extractant; (6) Add 95% ethanol to the secondary concentrate at an alcohol precipitation ratio of 1:3 while stirring, and then place it in a refrigerator at 5°C for alcohol precipitation overnight. (7) After the alcohol precipitation is completed, discard the supernatant, collect the precipitate and dry it until there is no alcohol smell.

[0030] The experiment used a fixed extraction time of 60 min, with a single extraction liquid to petroleum ether volume ratio of 2:3 and an alcohol precipitation ratio of 1:3. The results showed that the polysaccharide extraction rate increased with increasing solid-liquid ratio, reaching a maximum at a ratio of 1:60. Afterward, the polysaccharide extraction rate decreased with further increases in the solid-liquid ratio. This may be because under low solid-liquid ratio conditions, the extraction solvent is insufficient, and the polysaccharides in *Ophiopogon japonicus* cannot be fully dissolved, leading to a lower extraction rate. While a high solid-liquid ratio increases the extraction solvent, which is beneficial for polysaccharide dissolution, it also leads to a prolonged concentration process, causing some polysaccharide degradation and thus a decrease in extraction rate. The specific trend is as follows: Figure 3 As shown. Example 3

[0031] Single-factor experiment on the effect of extraction time on extraction rate This experiment aimed to investigate the effect of different extraction times on the extraction rate of Ophiopogon japonicus polysaccharides. The experimental steps are as follows: (1) Weigh 25g of dried Ophiopogon japonicus, add 40 times the amount of distilled water, and extract twice in an electromagnetic heating extractor. The extraction temperature is 100℃ under normal pressure, and the extraction time is set to 40 min, 50 min, 60 min, 70 min, and 80 min. After each extraction, filter twice with medical gauze while hot, and then filter twice with fine filter paper to remove the dregs and mud. Combine the extracts to obtain a mixed extract. (2) The mixed extract was concentrated to 12.5 ml using a rotary evaporator to obtain a first concentrate; (3) Add an equal volume of dichloromethane to the first concentrate, shake thoroughly to extract, let stand and separate the layers, discard the lower dichloromethane phase, retain the upper aqueous phase, and obtain the decolorized first extract. (4) The first extract was extracted with petroleum ether at a volume ratio of 2:3. After standing and separating the layers, the upper petroleum ether phase was discarded and the lower aqueous phase was retained to obtain the second extract in order to remove fat-soluble impurities. (5) Add distilled water to the secondary extract until the total volume is 25 ml, and concentrate it again to 12.5 ml to obtain a secondary concentrate to remove residual extractant; (6) Add 95% ethanol to the secondary concentrate at an alcohol precipitation ratio of 1:3 while stirring, and then place it in a refrigerator at 5°C for alcohol precipitation overnight. (7) After the alcohol precipitation is completed, discard the supernatant, collect the precipitate and dry it until there is no alcohol smell.

[0032] The experiment fixed the material-to-liquid ratio at 1:40, the volume ratio of the primary extract to petroleum ether at 2:3, and the alcohol precipitation ratio at 1:3. The results showed that the polysaccharide extraction rate increased with time within the extraction period of 40-70 min, reaching a maximum at 70 min. Afterward, further extension of the extraction time led to a decrease in the polysaccharide extraction rate. This may be because shorter extraction times result in insufficient polysaccharide dissolution, leading to a low extraction rate. Extending the extraction time can indirectly increase the dissolution time of polysaccharides. Conversely, prolonged extraction may cause polysaccharide molecules to boil inside the medicinal material, blocking diffusion channels and making it difficult for polysaccharides to dissolve, thus reducing the extraction rate. The specific trend is as follows: Figure 4 As shown. Example 4

[0033] Single-factor experiment on the effect of the volume ratio of primary extract to petroleum ether on extraction rate This experiment aimed to investigate the effect of the volume ratio of primary extract to petroleum ether on the extraction rate of Ophiopogon japonicus polysaccharides. The experimental steps are as follows: (1) Weigh 25g of dried Ophiopogon japonicus, add 40 times the amount of distilled water, and extract twice in an electromagnetic heating extractor. The extraction temperature is 100℃ under normal pressure, and each extraction lasts for 60 minutes. After each extraction, filter twice with medical gauze while hot, and then filter twice with fine filter paper to remove the dregs and mud. Combine the extracts to obtain a mixed extract. (2) The mixed extract was concentrated to 12.5 ml using a rotary evaporator to obtain a first concentrate; (3) Add an equal volume of dichloromethane to the primary concentrate, shake thoroughly to extract, let stand and separate the layers, discard the lower dichloromethane phase, and retain the upper aqueous phase to obtain the primary extract; (4) The primary extract is subjected to a secondary extraction with petroleum ether at a volume ratio (aqueous phase: petroleum ether = 2:1, 1:1, 1:2, 1:3, 1:4). After standing and separating the layers, the upper petroleum ether phase is discarded and the lower aqueous phase is retained to obtain the secondary extract, in order to remove fat-soluble impurities. (5) Add distilled water to the secondary extract until the total volume is 25 ml, and concentrate it again to 12.5 ml to obtain a secondary concentrate to remove residual extractant; (6) Add 95% ethanol to the secondary concentrate at an alcohol precipitation ratio of 1:3 while stirring, and then place it in a refrigerator at 5°C for alcohol precipitation overnight. (7) After the alcohol precipitation is completed, discard the supernatant, collect the precipitate and dry it until there is no alcohol smell.

[0034] The experiment fixed the solid-liquid ratio at 1:40, the extraction time at 60 min, and the alcohol precipitation ratio at 1:3. The results showed that as the volume ratio of petroleum ether increased, polysaccharide extraction initially increased and then decreased, reaching a maximum at a volume ratio of 2:1, then gradually decreasing, and stabilizing at a volume ratio of 1:3. This phenomenon may be related to the partitioning mechanism during extraction: when the petroleum ether ratio is low, appropriately increasing the volume of the organic phase helps disrupt cell structure, promoting the transfer of polysaccharides from the solid phase to the liquid phase, thus increasing the extraction rate; however, when the petroleum ether ratio is too high, the volume of the aqueous phase decreases relatively, leading to a decrease in the concentration of polysaccharides in the aqueous phase, inhibiting their partitioning to the organic phase, and causing the extraction rate to decrease; when the volume ratio further increases to 1:3, the partitioning equilibrium between the two phases gradually approaches saturation, the change in extraction rate slows down, and finally stabilizes, as shown in the following trend. Figure 5 As shown. Example 5

[0035] Single-factor experiment on the effect of alcohol precipitation ratio on extraction rate This experiment aimed to investigate the effect of alcohol precipitation ratio on the extraction rate of Ophiopogon japonicus polysaccharides. The experimental steps are as follows: (1) Weigh 25g of dried Ophiopogon japonicus, add 40 times the amount of distilled water, and extract twice in an electromagnetic heating extractor. The extraction temperature is 100℃ under normal pressure, and each extraction lasts for 60 minutes. After each extraction, filter twice with medical gauze while hot, and then filter twice with fine filter paper to remove the dregs and mud. Combine the extracts to obtain a mixed extract. (2) The mixed extract was concentrated to 12.5 ml using a rotary evaporator to obtain a first concentrate; (3) Add an equal volume of dichloromethane to the first concentrate, shake thoroughly to extract, let stand and separate the layers, discard the lower dichloromethane phase, retain the upper aqueous phase, and obtain the decolorized first extract. (4) The first extract was extracted with petroleum ether at a volume ratio of 2:3. After standing and separating the layers, the upper petroleum ether phase was discarded and the lower aqueous phase was retained to obtain the second extract in order to remove fat-soluble impurities. (5) Add distilled water to the secondary extract until the total volume is 25 ml, and concentrate it again to 12.5 ml to obtain a secondary concentrate to remove residual extractant; (6) Add 95% ethanol to the secondary concentrate at the ethanol precipitation ratio (1:3, 1:3.5, 1:4, 1:4.5, 1:5) while stirring, and then place it in a refrigerator at 5°C for ethanol precipitation overnight. (7) After the alcohol precipitation is completed, discard the supernatant, collect the precipitate and dry it until there is no alcohol smell.

[0036] The experiment fixed the material-to-liquid ratio at 1:40, the extraction time at 60 min, and the petroleum ether volume ratio at 2:3. The results showed that within the alcohol-to-precipitation ratio range of 1:3-1:4, the polysaccharide extraction rate increased with increasing alcohol-to-precipitation ratio, reaching its maximum at 1:4. With further increases in the alcohol-to-precipitation ratio, the polysaccharide extraction rate decreased. This may be because the increased ethanol content in the precipitate reduced the dielectric constant of the system, decreasing the stability of the bond between water molecules and the hydroxyl groups of the sugars. This caused the hydroxyl groups on the polysaccharide molecular chains to become entangled, making it easier to form stable hydrogen bonds and resulting in tighter entanglement and easier precipitation. Alternatively, increasing the ethanol dosage, within a certain range, reduced the solubility of the polysaccharides, which was beneficial for precipitation. However, further increases in ethanol dosage caused some impurities to precipitate, which was detrimental to polysaccharide precipitation, thus reducing the extraction rate. The specific trend is as follows: Figure 6 As shown. Example 6

[0037] Orthogonal experiment for extracting polysaccharides from Ophiopogon japonicus The specific process is as follows: Through single-factor analysis, the L9(3) model was designed by selecting the material-to-liquid ratio, extraction time, and alcohol precipitation ratio. 4Orthogonal experiments were conducted to optimize the extraction process, and the levels of each factor are shown in Table 1. Considering the combined effects of extract volume and volume ratio on the extraction rate, a volume ratio of 2:1 was adopted to ensure the highest polysaccharide extraction rate while using low petroleum ether dosage. The orthogonal experimental data (Table 2) were processed and analyzed using SPSS 24.0, yielding range analysis (Table 3) and factor mean values ​​plots (…). Figure 7 ) and analysis of variance (Table 4).

[0038] Table 1 Orthogonal Factor Level Table .

[0039] Table 2 Orthogonal Experiment Data .

[0040] Table 3 Range Analysis .

[0041] Table 4 Analysis of Variance .

[0042] As shown in Table 4, the effects of the material-to-liquid ratio, extraction temperature, and alcohol precipitation ratio on the extraction rate of Ophiopogon japonicus polysaccharides were all statistically significant (P<0.05); according to Table 3, the range R:R A >R C >R B The order of influence of various factors on the extraction rate of wheat polysaccharides is as follows: material-to-liquid ratio > alcohol precipitation ratio > extraction time; (Based on Table 3 and...) Figure 7 The optimal extraction process parameters were determined to be A1B3C2, namely, a material-to-liquid ratio of 1:55, an extraction time of 75 min, and an alcohol precipitation ratio of 1:4. These process conditions are consistent with those in Example 1.

[0043] Three parallel experiments were conducted under the optimal extraction process parameters to verify the optimal extraction process parameters. The results are shown in Table 5.

[0044] Table 5. Validation results of orthogonal optimal extraction process .

[0045] As shown in Table 5, the optimal extraction process for Ophiopogon japonicus polysaccharide yielded an extraction rate of 51.74%, and the RSD of three extractions was 1.11%, indicating that the process is stable and feasible.

[0046] Example 7 Methodological validation of Ophiopogon japonicus polysaccharide extraction process 1. Precision Experiment Prepare a 0.1 mg / mL glucose standard solution, then sonicate the glucose standard solution for 20 min and let it stand for 1.5 h. Accurately pipette 9 portions of the 0.1 mg / mL reference solution, measure the absorbance value A, and calculate the RSD value to verify whether the instrument precision meets the requirements. The experimental results are shown in Table 6.

[0047] Table 6 Precision Experiment Results .

[0048] As shown in Table 6, RSD = 1.23% (RSD < 2%), indicating that the instrument has good precision.

[0049] 2. Stability test The polysaccharide obtained under the optimal process conditions was prepared into a polysaccharide test sample. The test sample was sonicated for 20 min and then allowed to stand for 1.5 h. Then, 8 samples of 1 ml polysaccharide test sample were accurately pipetted and the absorbance value A was measured at 0, 2, 4, 6, 8, 10, 12 and 24 h. The RSD value was calculated to examine whether the stability of the sample solution was good. The results are shown in Table 7.

[0050] Table 7 Stability test results .

[0051] As shown in Table 7, RSD = 1.69% (RSD < 2%), indicating that the polysaccharide solution has good stability within 24 hours.

[0052] 3. Reproducibility test Six 1ml aliquots of polysaccharide test sample were accurately pipetted, and the absorbance value A was measured. The RSD value was calculated to verify the reproducibility of this method for determining polysaccharide content. The results are shown in Table 8.

[0053] Table 8 Reproducibility Test Results .

[0054] As shown in Table 8, the RSD is 1.13% (RSD < 2%), indicating that the method has good consistency in determining polysaccharide content.

[0055] 4. Spiking recovery experiment First, accurately pipette three 0.5 mL aliquots of the test solution, then add 0.5 mL of distilled water to make up to 1 mL, and measure the absorbance value A. Then, accurately pipette nine 0.5 mL aliquots of the test solution, divide them into three groups, and accurately add 0.5 mL of 80%, 100%, and 120% 0.1 mg / mL grape standard reference solution to each group, respectively, and measure the absorbance value A. Calculate the average recovery rate and RSD. The results are shown in Table 9.

[0056] Table 9 Results of the spiking recovery experiment

[0057] Table 9 shows that the recoveries (P) for each group were 99.05%, 101.46%, and 99.31%, with an average recovery rate of 99.94%. The fact that the RSD < 2% indicates that this method has a good recovery rate.

[0058] Comparative Example 1 This comparative study investigated the effect of reflux extraction on the extraction of polysaccharides from Ophiopogon japonicus, as detailed below: (1) Weigh 25g of dried Ophiopogon japonicus, add 55 times the amount of water and place it in a 1000 mL round bottom flask. Reflux and extract twice, 75 min each time. After each extraction, filter twice with medical gauze while hot, and then filter twice with fine filter paper to remove the dregs and mud. Combine the extracts to obtain a mixed extract. (2) The mixed extract was concentrated to 12.5 ml using a rotary evaporator to obtain a first concentrate; (3) Add an equal volume of dichloromethane to the first concentrate, shake thoroughly to extract, let stand and separate the layers, discard the lower dichloromethane phase, retain the upper aqueous phase, and obtain the decolorized first extract. (4) The primary extract was extracted with petroleum ether at a volume ratio of 2:1. After standing and separating the layers, the upper petroleum ether phase was discarded and the lower aqueous phase was retained to obtain the secondary extract in order to remove fat-soluble impurities. (5) Add distilled water to the secondary extract until the total volume is 25 ml, and concentrate it again to 12.5 ml to obtain a secondary concentrate to remove residual extractant; (6) Add 95% ethanol to the secondary concentrate at an alcohol precipitation ratio of 1:4 while stirring, and then place it in a refrigerator at 5°C for alcohol precipitation overnight. (7) After the alcohol precipitation is completed, discard the supernatant, collect the precipitate and dry it until there is no alcohol smell.

[0059] Comparative Example 2 This comparative study investigated the effectiveness of ultrasonic extraction for extracting polysaccharides from Ophiopogon japonicus, as detailed below: 1) Weigh 25g of dried Ophiopogon japonicus, add 55 times the amount of water, and extract twice under ultrasonic conditions of 40KHz (extraction mode: ultrasonic for 30min, interval of 20min, cycle 5 times), each time for 75min. After each extraction, filter twice with medical gauze while hot, and then filter twice with fine filter paper to remove the dregs and mud. Combine the extracts to obtain a mixed extract. (2) The mixed extract was concentrated to 12.5 ml using a rotary evaporator to obtain a first concentrate; (3) Add an equal volume of dichloromethane to the first concentrate, shake thoroughly to extract, let stand and separate the layers, discard the lower dichloromethane phase, retain the upper aqueous phase, and obtain the decolorized first extract. (4) The primary extract was extracted with petroleum ether at a volume ratio of 2:1. After standing and separating the layers, the upper petroleum ether phase was discarded and the lower aqueous phase was retained to obtain the secondary extract in order to remove fat-soluble impurities. (5) Add distilled water to the secondary extract until the total volume is 25 ml, and concentrate it again to 12.5 ml to obtain a secondary concentrate to remove residual extractant; (6) Add 95% ethanol to the secondary concentrate at an alcohol precipitation ratio of 1:4 while stirring, and then place it in a refrigerator at 5°C for alcohol precipitation overnight. (7) After the alcohol precipitation is completed, discard the supernatant, collect the precipitate and dry it until there is no alcohol smell.

[0060] Three parallel experiments were conducted to compare the extraction method of Example 1, the reflux extraction method, and the ultrasonic extraction method. Each method was independently repeated three times. The results showed that the extraction method of the present invention has a significant effect on improving the extraction efficiency of Ophiopogon japonicus polysaccharide. Specific data are shown in Table 10. Table 10 Effect of different extraction methods on the extraction rate of Ophiopogon japonicus polysaccharides .

[0061] Table 10 shows that the average RSD of the three extraction methods is less than 2%, indicating that each extraction method is stable and feasible. The extraction rates of Ophiopogon japonicus polysaccharides from reflux extraction, ultrasonic extraction, and extraction in Example 1 were 26.67%, 40.70%, and 51.74%, respectively. The extraction rate of Ophiopogon japonicus polysaccharides from Example 1 was 1.94 times that of reflux extraction and 11.04% higher than that of ultrasonic extraction. The results indicate that the extraction technology of the present invention can significantly improve the extraction rate of Ophiopogon japonicus polysaccharides and has obvious advantages over reflux extraction and ultrasonic extraction. Ultrasonic extraction also has certain advantages, with an extraction rate higher than that of reflux extraction. Ultrasonic extraction outperforms reflux extraction, likely due to the cavitation, mechanical, and thermal effects generated by ultrasound at certain frequencies. Cavitation creates numerous tiny bubbles within the liquid within moments; the high temperature and pressure generated when these bubbles burst loosen tightly bound plant tissues and even damage cell walls, facilitating the dissolution of intercellular and intracellular polysaccharides. The mechanical effect causes water particles to vibrate in space, enhancing the propagation and diffusion of the water, which in turn promotes polysaccharide diffusion and dissolution. The thermal effect facilitates rapid energy transfer between the water and polysaccharides, increasing the dissolution rate. The combined effect of these three effects promotes the release, dissolution, and diffusion of polysaccharide components, allowing them to transfer more and faster into distilled water, thus increasing the polysaccharide extraction rate.

[0062] The reason why the extraction efficiency of this invention is higher than that of ultrasonic extraction may be as follows: Electromagnetic heating extraction is used. An alternating magnetic field is generated by an electric current passing through a coil. Under this alternating magnetic field, eddy currents are generated in the magnetically conductive reactor. This causes the originally disordered polar molecules inside the material to align in an orderly manner according to a certain orientation under the influence of electromagnetic waves generated by the high-frequency alternating magnetic field. During this continuous change in orientation, the molecules move and collide, converting the field energy of the electromagnetic field into the thermal energy of water. This rapidly increases the temperature inside the cell, causing the intracellular liquid water to vaporize. This leads to a continuous increase in intracellular pressure until it exceeds the maximum pressure that the cell wall can withstand, causing it to rupture and form micropores. Simultaneously, the increased temperature further reduces the vaporization of water in the cell and cell wall, leading to cell contraction and the formation of cracks. These micropores and cracks promote the exchange of substances between the inside and outside of the cell, accelerating the entry of solvent into the cell and the dissolution and release of polysaccharides into the extracellular space, thus facilitating the extraction and dissolution of polysaccharides.

[0063] Furthermore, electromagnetic heating extraction utilizes non-contact high-frequency electromagnetic field heating technology to achieve a heat conversion rate exceeding 90%, increasing heating speed by 60% compared to traditional condensation extraction and ultrasonic extraction, significantly shortening preheating time, and reducing energy consumption by over 30%. This high efficiency can significantly improve the conversion rate of polysaccharides from traditional Chinese medicine, providing crucial data support for the research and development of polysaccharide preparations. Simultaneously, leveraging its core advantages of energy saving, high efficiency, and environmental friendliness, it promotes the transformation of the traditional Chinese medicine preparation industry towards a sustainable development model characterized by innovation, energy conservation, emission reduction, and long-term operation.

Claims

1. A method for extracting polysaccharides from Ophiopogon japonicus, characterized in that, Includes the following steps: (1) Mix Ophiopogon japonicus with distilled water at a material-liquid ratio of 1:40-70 and extract twice under normal pressure in an electromagnetic heating extractor. The extraction temperature is 100℃ and the extraction time is 60-80min each time. After each extraction, filter twice with medical gauze while hot, and then filter twice with fine filter paper. Combine the extracts to obtain a mixed extract. (2) The mixed extract was concentrated using a rotary evaporator until the dry weight volume ratio of Ophiopogon japonicus was 2:1 to obtain a first-concentrated solution; (3) Add an equal volume of dichloromethane to the primary concentrate, shake thoroughly to extract, let stand and separate the layers, discard the lower dichloromethane phase, retain the upper aqueous phase, and obtain the decolorized primary extract. (4) The primary extract was extracted with petroleum ether at a volume ratio of 2.5:1 to 1:

1. After standing and separating the layers, the upper petroleum ether phase was discarded and the lower aqueous phase was retained to obtain the secondary extract. (5) Add an appropriate amount of distilled water to the secondary extract and concentrate it again until the dry weight volume ratio of Ophiopogon japonicus is 2:1 to obtain the secondary concentrate. (6) Add 95% ethanol to the secondary concentrate at an alcohol precipitation ratio of 1:3-1:4.5 while stirring, and then place it in a refrigerator at 5°C for alcohol precipitation overnight; (7) After the alcohol precipitation is completed, discard the supernatant, collect the precipitate and dry it until there is no alcohol smell.

2. The method for extracting Ophiopogon japonicus polysaccharides according to claim 1, characterized in that, In step (1), the material-to-liquid ratio is 1:

55.

3. The method for extracting Ophiopogon japonicus polysaccharides according to claim 1, characterized in that, The extraction time in step (1) is 75 minutes each time.

4. The method for extracting Ophiopogon japonicus polysaccharides according to claim 1, characterized in that, The volume ratio in step (4) is 2:

1.

5. The method for extracting Ophiopogon japonicus polysaccharides according to claim 1, characterized in that, In step (6), the alcohol precipitation ratio is 1:

4.

6. The method for extracting Ophiopogon japonicus polysaccharides according to claim 1, characterized in that, The electromagnetic heating extractor is model YJDJ08.

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