Method for ultrasonic-assisted deep eutectic solvent extraction of polysaccharides from gastrodia elata blume
Patent Information
- Application Number
- CN202611293293.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-08-25
- Publication Date
- 2026-10-20
AI Technical Summary
该工艺存在诸多技术缺陷：一是多糖提取得率极低，仅为7%~8%，原料有效成分利用率低，物料损耗严重；二是长期高温浸提环境会断裂多糖糖苷键，造成大分子多糖降解，严重破坏天麻多糖抗氧化、免疫调节等核心生物活性；三是工艺浸提耗时长、重复操作工序烦琐，整体能耗高、提取周期长，生产效率低下；四是纯水对天麻致密的木质纤维素细胞壁渗透能力有限，无法充分溶出胞内多糖；五是提取废水无回收利用价值，后续废液处理难度大
[0039]1、多糖提取得率大幅提升：最优工艺下昭通乌天麻多糖得率稳定达21.05%；传统热水浸提法仅7.66%，相对提升率计算公式为(21.05-7.66)÷7.66×100%=174.8%，同等原料下多糖产量提升近1.75倍；单一DES静态浸泡最高得率19.75%，耦合超声后得率额外提升6.6%，原料利用率显著提高；
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of natural product active ingredient extraction technology, specifically relating to a method for extracting polysaccharides from Gastrodia elata using ultrasound-assisted eutectic solvent. Background Technology
[0002] Gastrodia elata polysaccharides are the core functional active substances in Gastrodia elata, possessing various biological activities such as anti-oxidation, anti-aging, immunomodulation, neuroprotection, anti-inflammatory and antibacterial effects, improvement of cardiovascular function, and regulation of intestinal flora. They are a key research focus in the field of natural product development. The efficient, complete, and stable acquisition of highly active Gastrodia elata polysaccharides is the core research objective of Gastrodia elata deep processing technology. Existing Gastrodia elata polysaccharide extraction processes have inherent technical defects, generally suffering from low polysaccharide extraction rates, severe activity degradation, low extraction efficiency, poor process stability, and difficult post-processing, which greatly limits the development and application of efficient Gastrodia elata polysaccharide preparation technology. Therefore, developing an efficient, low-loss, and scalable Gastrodia elata polysaccharide extraction process has significant technical research value. Currently, the mainstream extraction processes for Gastrodia elata polysaccharides are mainly traditional hot water extraction, single enzyme-assisted extraction, and single eutectic solvent static immersion extraction. Each of these single processes has unavoidable technical shortcomings, making it difficult to simultaneously meet the technical requirements of high extraction rate, high activity, high efficiency, low cost, and easy industrialization. Specific existing technical solutions and their inherent defects are as follows.
[0003] The existing technology is the traditional hot water extraction method, which is the conventional basic process for extracting Gastrodia elata polysaccharides. This technology uses deionized water as the sole extraction solvent. After the Gastrodia elata raw material is crushed and sieved, it is continuously stirred and extracted in a high-temperature constant temperature water bath at 80~100℃. The extraction time for each extraction is 2~4 hours. The filtrates are combined through multiple extractions, and then crude Gastrodia elata polysaccharides are obtained through processes such as ethanol precipitation, centrifugation, dialysis to remove impurities, and vacuum drying. The material-to-liquid ratio is controlled at 1:30~1:60 (g:mL) in the conventional process, and no external force is used to assist in crushing and mass transfer enhancement. This process has several technical drawbacks: First, the polysaccharide extraction yield is extremely low, only 7% to 8%, resulting in low utilization of the effective components of the raw materials and significant material loss. Second, the long-term high-temperature extraction environment breaks the glycosidic bonds of polysaccharides, causing the degradation of macromolecular polysaccharides and severely damaging the core biological activities of Gastrodia elata polysaccharides, such as antioxidant and immunomodulatory effects. Third, the extraction process is time-consuming, involves cumbersome repetitive operations, has high overall energy consumption, a long extraction cycle, and low production efficiency. Fourth, pure water has limited penetration into the dense lignocellulose cell walls of Gastrodia elata, making it impossible to fully dissolve the intracellular polysaccharides. Fifth, the extraction wastewater has no recycling value, and subsequent waste liquid treatment is difficult.
[0004] Existing technology two is a single enzyme-assisted extraction method. This process uses enzymatic hydrolysis to break down the cell walls of Gastrodia elata to enhance the polysaccharide dissolution effect. The technical solution involves using a compound enzyme preparation including cellulase, α-amylase, and pectinase to perform isothermal enzymatic hydrolysis of Gastrodia elata powder under a specific temperature and pH buffer system. The enzymes rely on their specific hydrolytic action to decompose the cell wall cellulose and hemicellulose skeleton, releasing intracellular polysaccharides. After enzymatic hydrolysis, the polysaccharides are prepared through processes such as high-temperature enzyme inactivation, filtration, alcohol precipitation, deproteinization, and dialysis drying. The technical drawbacks of this process are quite prominent: First, commercially available compound enzyme preparations are expensive and not suitable for large-scale continuous production; second, the enzymatic hydrolysis reaction is highly sensitive to temperature, pH, and substrate concentration, with a narrow operating window, resulting in poor process stability and repeatability after industrial scale-up; third, the enzymatic hydrolysis system leaves behind a large amount of denatured proteins and unreacted enzyme proteins, significantly increasing the difficulty of subsequent deproteinization and purification, and causing loss of active polysaccharide materials; fourth, relying solely on enzymatic hydrolysis for mass transfer without physical disruption assistance, the improvement in polysaccharide yield is limited, and the optimization effect is not obvious; fifth, the enzymatic hydrolysis waste liquid is rich in organic proteins, resulting in high costs for subsequent environmental treatment.
[0005] The third existing technology is a static immersion extraction method using a single eutectic solvent. This process uses a novel green solvent to replace traditional pure water and organic solvents for polysaccharide extraction. The technical solution is as follows: a eutectic solvent is prepared by selecting conventional hydrogen bond donor-acceptor combinations such as choline chloride-ethylene glycol, choline chloride-urea, and choline chloride-oxalic acid. Gastrodia elata powder is mixed with the eutectic solvent and then statically immersed in a water bath at a constant temperature. No external mass transfer enhancement methods such as ultrasound or stirring are used. After immersion, conventional purification processes such as solid-liquid separation, alcohol precipitation, and drying are performed to obtain Gastrodia elata polysaccharides. This process has significant technical shortcomings: First, the eutectic solvent itself has a high viscosity, and without external force assistance in the static immersion mode, the system experiences high mass transfer resistance, resulting in a slow polysaccharide dissolution rate and a single immersion time of 2-3 hours. Second, relying solely on solvent hydrogen bonding for extraction cannot effectively disrupt the dense cell wall structure of Gastrodia elata, leading to insufficient release of intracellular polysaccharides. Third, the general eutectic solvent system has not been specifically screened and adapted for the Gastrodia elata raw material matrix, and most solvents have weak dissolution and extraction capabilities for Gastrodia elata polysaccharides. Fourth, experimental verification shows that under the optimal conventional eutectic solvent static extraction process, the highest yield of Gastrodia elata polysaccharides is only 19.75%, indicating significant room for improvement in extraction efficiency.
[0006] In summary, existing single extraction processes all have significant technical shortcomings. Hot water extraction results in poor polysaccharide activity and low yield; enzyme-assisted extraction suffers from poor process stability and high cost; and static extraction using a single eutectic solvent has low mass transfer efficiency and incomplete extraction. Furthermore, current technologies rarely couple and synergistically utilize ultrasound-assisted physical extraction with eutectic solvent extraction for the targeted extraction of Gastrodia elata polysaccharides. There is a lack of a dedicated eutectic solvent formulation system adapted to the Gastrodia elata matrix, and no integrated extraction process with controllable parameters, excellent repeatability, and industrial scalability. Therefore, there is an urgent need to develop an ultrasound-assisted eutectic solvent coupled extraction process that combines the advantages of physical disruption to enhance mass transfer with efficient extraction using green solvents, enabling large-scale extraction of Gastrodia elata polysaccharides with high yield, high activity, and high efficiency. Summary of the Invention
[0007] In view of the above shortcomings, this invention provides a green and efficient process for extracting polysaccharides from *Gastrodia elata* var. *zhaotongensis* using an ultrasound-assisted eutectic solvent, comprising: a raw material pretreatment unit, preparation of a specific eutectic solvent, ultrasound-coupled extraction, polysaccharide purification, and quantitative detection of polysaccharides. To solve the above technical problems, this invention adopts the following technical means:
[0008] This invention first discloses a method for extracting polysaccharides from Gastrodia elata using an ultrasound-assisted eutectic solvent, comprising the following steps:
[0009] (1) Raw material pretreatment
[0010] Mature Gastrodia elata tubers from Zhaotong, Yunnan Province were selected and dried at a constant temperature of 45℃ in an electric heating forced-air drying oven until constant weight was achieved. The constant weight determination standard was that the difference in mass between two weighings of Gastrodia elata tubers at 2-hour intervals should not exceed 0.5 mg, and the moisture content of the dried Gastrodia elata raw material should be controlled below 5%. The dried Gastrodia elata tubers were crushed using a pulverizer and all were passed through a 40-mesh standard sieve. The sieved Gastrodia elata powder was sealed and stored at room temperature away from light for later use.
[0011] (2) Preparation of DES in eutectic solvent
[0012] Raw material ratio: Quaternary ammonium salt hydrogen bond acceptors and oxygen-containing organic complexing agents hydrogen bond donors, with a molar ratio strictly controlled at 1:2 to 2:1; add 20% to 50% deionized water by mass of the total solvent.
[0013] Synthesis procedure: Accurately weigh the quaternary ammonium salt hydrogen bond acceptor and the oxygen-containing organic complexing agent hydrogen bond donor, add the measured amount of deionized water and mix, place in an 80°C constant temperature water bath, and continuously stir with a magnetic force until the system forms a clear, transparent, homogeneous, non-layered, and non-crystallized viscous liquid.
[0014] Post-processing: Stop heating and allow to cool naturally to room temperature to obtain a special DES extraction solvent suitable for extracting polysaccharides from Gastrodia elata in Zhaotong.
[0015] Furthermore, the quaternary ammonium salt hydrogen bond acceptors include, but are not limited to, choline chloride and betaine.
[0016] Furthermore, the quaternary ammonium salt hydrogen bond acceptor is choline chloride.
[0017] Furthermore, the oxygen-containing organic complexing agent includes, but is not limited to: DL-malic acid, citric acid, lactic acid, ethylene glycol, 1,2-propanediol, glycerol, glucose, fructose, glacial acetic acid, and oxalic acid.
[0018] Furthermore, the oxygen-containing organic complexing agent is DL-malic acid; and the molar ratio of choline chloride to DL-malic acid is 1:1.
[0019] Furthermore, the addition of deionized water accounts for 30% of the total mass of the solvent.
[0020] (3) Ultrasound-assisted DES extraction of Gastrodia elata polysaccharide
[0021] Feeding ratio: Accurately weigh the pretreated Gastrodia elata powder, add the prepared DES solvent according to the material-liquid ratio of 1:30~50 (g:mL), and stir thoroughly to mix well;
[0022] Ultrasonic extraction parameters: Place the mixture in a tank-type ultrasonic cleaner (or a probe-type ultrasonic processor or an industrial-grade ultrasonic extraction tank to achieve uniform distribution of the ultrasonic field during scale-up production), fix the ultrasonic power at 100~300W, and maintain a constant temperature of 35~55℃ for 35~55min.
[0023] Solid-liquid separation: After extraction, the material is removed and allowed to cool naturally to room temperature; centrifuge at 5000 r / min for 10 min or vacuum filter to remove solid residues of Gastrodia elata and collect the clear crude polysaccharide extract.
[0024] Furthermore, the ratio of the care solution is 1:50 (g:mL).
[0025] Furthermore, the fixed ultrasonic power is 180W, and the constant temperature is 45℃ for continuous ultrasonic treatment for 45 minutes.
[0026] (4) Complete purification process of Gastrodia elata polysaccharide
[0027] First alcohol precipitation: Add 4 times the volume of anhydrous ethanol to the crude extract, add slowly while stirring, mix evenly, and then transfer to a 4°C refrigerated environment for alcohol precipitation for 12 hours.
[0028] Precipitation collection: Centrifuge at 5000 r / min for 10 min and collect the white polysaccharide precipitate from the bottom of the test tube;
[0029] Reconstitution and deproteinization: The polysaccharide precipitate was fully reconstituted in deionized water. Deproteinization reagent was added at a ratio of polysaccharide solution to Sevage reagent of 4:1 (V / V). Sevage reagent was prepared by mixing chloroform and n-butanol at a volume ratio of 4:1. After mixing, the mixture was vigorously shaken for 20 min, centrifuged at 7500 r / min for 25 min, and the layers were separated. The lower organic phase and the intermediate denatured protein layer were removed. The deproteinization process was repeated 3 to 5 times until there was no visible white flocculent protein precipitate at the interface between the two phases, and no characteristic protein absorption peak was detected at 280 nm by ultraviolet spectrophotometry.
[0030] Secondary alcohol precipitation dialysis: After deproteinization, the polysaccharide aqueous solution was added again with 4 times the volume of anhydrous ethanol and refrigerated at 4°C overnight for alcohol precipitation; after centrifugation to collect the precipitate, the polysaccharide precipitate was dissolved in deionized water, placed in a dialysis bag with a molecular weight cutoff of 3500 Da, and dialyzed with distilled water for 48 hours. The distilled water was changed every 8-12 hours during the dialysis process to completely remove DES small molecule impurities.
[0031] Drying and shaping: After dialysis, the polysaccharide aqueous solution is transferred to a freeze dryer and freeze-dried at low temperature to obtain pure white high-purity Gastrodia elata polysaccharide powder.
[0032] (5) Validation method for quantitative detection of polysaccharides (phenol-sulfuric acid method)
[0033] This method is used to verify the polysaccharide extraction effect of this process. It is not part of the extraction procedure, but it serves as a standard method for quantitatively verifying the process effect.
[0034] Reagent preparation: Prepare a 5% (w / w) aqueous solution of phenol, and prepare a gradient standard solution using D-glucose as the standard;
[0035] Colorimetric detection: Take 1 mL of the polysaccharide solution to be tested, add 1 mL of 5% phenol solution and 5 mL of concentrated sulfuric acid in sequence, shake to mix, and then incubate at 45℃ for 25 min for color development. Cool to room temperature. Use pure water as a blank control group. Measure the absorbance at 490 nm wavelength with a UV spectrophotometer. Measure each sample in parallel 3 times.
[0036] Standard curve: A standard curve was plotted with the glucose standard concentration C (mg / mL) on the ordinate and the measured absorbance A at 490 nm on the abscissa. Linear regression was then performed, yielding the regression equation: C = 0.0969A + 0.0003, R0 2 =0.9993 where C is the polysaccharide concentration (mg / mL) and A is the measured absorbance value at a wavelength of 490 nm;
[0037] The formula for calculating the yield is: Gastrodia elata polysaccharide yield (%) = (C × V × N / m) × 100% Where: C is the polysaccharide concentration of the test solution (mg / mL); V is the volume of the test solution (mL); N is the dilution factor of the solution; and m is the mass of Gastrodia elata powder raw material (g).
[0038] The beneficial effects of this invention are as follows:
[0039] 1. Significantly improved polysaccharide extraction yield: Under the optimal process, the polysaccharide yield of Gastrodia elata from Zhaotong reached a stable 21.05%; the traditional hot water extraction method yielded only 7.66%. The relative improvement rate calculation formula is (21.05-7.66)÷7.66×100%=174.8%, which means that the polysaccharide yield increased by nearly 1.75 times under the same raw materials; the highest yield of single DES static soaking was 19.75%, and the yield was further increased by 6.6% after coupling with ultrasound, significantly improving the utilization rate of raw materials.
[0040] 2. Mild extraction conditions preserve the full bioactivity of polysaccharides: The extraction temperature is only 45℃, which is much lower than the high temperature of 80~100℃ for hot water extraction. This effectively avoids the high-temperature breakage of polysaccharide glycosidic bonds and fully preserves the core activities of polysaccharides such as antioxidant and immunomodulatory effects. No acid, alkali or biological enzymes are required, and there is no risk of polysaccharide degradation.
[0041] 3. The extraction cycle is greatly shortened and energy consumption is significantly reduced: a single ultrasonic extraction takes only 45 minutes, while hot water extraction takes 2-4 hours per extraction. Under the same production capacity, the overall energy consumption is reduced by more than 70%, and the production efficiency is increased by 3-5 times.
[0042] 4. The solvent is green, environmentally friendly and biodegradable: choline chloride-DL malic acid DES has no volatile toxic organic solvents, can be completely biodegradable, and there is no discharge of highly polluting acid or alkali waste liquid during the entire extraction process. The post-treatment is simple and the environmental protection cost is extremely low.
[0043] 5. Stable process and easy industrial scale-up: Orthogonal experiments and three sets of parallel verification experiments have proven that the four parameters of material-liquid ratio, water content, ultrasonic time and ultrasonic temperature are controllable in a coordinated manner. The polysaccharide yield in repeated experiments is stable in the range of 20.8%~21.1%, and the industrial continuous production has good repeatability.
[0044] 6. Raw materials are specifically adapted to local Zhaotong Gastrodia elata: The DES system is specially screened for cell wall components and polysaccharide dissolution characteristics of Zhaotong Gastrodia elata. Compared with general plant extraction solvents, it has stronger specificity for breaking down the cell wall of Gastrodia elata matrix and dissolving polysaccharides, making it suitable for the deep processing industry of Chinese medicinal materials in Zhaotong.
[0045] 7. Simplified purification process and low material loss: No complex enzymes are required, no large amount of denatured protein impurities are generated, the deproteinization operation is repeated less often, and the polysaccharide purification recovery rate is higher.
[0046] 8. The extraction method and DES composition of this invention can also be applied to the polysaccharide extraction of other Gastrodia elata variants (such as red Gastrodia elata and green Gastrodia elata) or other medicinal and edible herbs rich in polysaccharides (such as Astragalus membranaceus, Ganoderma lucidum, and Poria cocos), but Zhaotong black Gastrodia elata is the most preferred raw material. This invention protects the application of this process in the deep processing of Zhaotong black Gastrodia elata, the preparation of functional foods, and Gastrodia elata health products. Attached Figure Description
[0047] Figure 1 The effect of different DES on the yield of Gastrodia elata polysaccharides. Detailed Implementation
[0048] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0049] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0050] The following are abbreviations and related meanings of the proprietary terms involved in this invention:
[0051] DES: Deep Eutectic Solvent, a low-melting-point, transparent, viscous, homogeneous liquid formed by heating and mixing hydrogen bond acceptor choline chloride and hydrogen bond donor in a fixed molar ratio. It has the characteristics of being biodegradable and having a strong ability to dissolve polar macromolecules.
[0052] UAE: Ultrasound-Assisted Extraction is a green extraction technology that relies on the triple action of ultrasonic cavitation, mechanical oscillation and local thermal effect to break down plant cell walls and enhance the mass transfer and dissolution of intracellular active substances.
[0053] Sevage method: A deproteinizing reagent prepared by chloroform and n-butanol in a volume ratio of 4:1 is used as a purification method to remove free impurities from crude polysaccharide extracts.
[0054] Phenol-sulfuric acid method: A standard detection method for quantitative determination of polysaccharide content using D-glucose as a standard and ultraviolet spectrophotometry at 490 nm.
[0055] Zhaotong Black Gastrodia: Specifically refers to a variant of Gastrodia elata produced in Zhaotong, Yunnan Province, with the scientific name Gastrodia elata Bl.f.glauca S.Chow (the italicized parts are the genus name, specific epithet, and variant epithet; these must be italicized in official printed documents). It is distinct from green and red Gastrodia elata. Zhaotong Black Gastrodia elata has thick tubers and significantly higher accumulations of gastrodin and gastrodia polysaccharides than Gastrodia elata from other producing areas, making it a unique and suitable raw material for the process of this invention.
[0056] Example 1
[0057] A method for extracting polysaccharides from Gastrodia elata using an ultrasound-assisted eutectic solvent includes the following steps:
[0058] (1) Raw material pretreatment
[0059] Mature Gastrodia elata tubers from Zhaotong, Yunnan Province were selected and dried at a constant temperature of 45℃ in an electric heating forced-air drying oven until constant weight was achieved. The constant weight determination standard was that the difference in mass between two weighings of Gastrodia elata tubers at 2-hour intervals should not exceed 0.5 mg, and the moisture content of the dried Gastrodia elata raw material should be controlled below 5%. The dried Gastrodia elata tubers were crushed using a pulverizer and all were passed through a 40-mesh standard sieve. The sieved Gastrodia elata powder was sealed and stored at room temperature away from light for later use.
[0060] (2) Preparation of DES in eutectic solvent
[0061] Raw material ratio: choline chloride and DL-malic acid, with a strictly controlled molar ratio of 1:1; add 30% deionized water by mass of the total solvent.
[0062] Synthesis procedure: Accurately weigh choline chloride and DL-malic acid, add metered deionized water and mix, place in an 80℃ constant temperature water bath, and continuously stir with a magnetic force until the system forms a clear, transparent, homogeneous, non-layered, and non-crystallized viscous liquid.
[0063] Post-processing: Stop heating and allow to cool naturally to room temperature to obtain a special DES extraction solvent suitable for extracting polysaccharides from Gastrodia elata in Zhaotong.
[0064] (3) Ultrasound-assisted DES extraction of Gastrodia elata polysaccharide
[0065] Feeding ratio: Accurately weigh the pretreated Gastrodia elata powder, add the prepared DES solvent according to the material-liquid ratio of 1:50 (g:mL), and stir thoroughly to mix well;
[0066] Ultrasonic extraction parameters: Place the mixture in a tank-type ultrasonic cleaner, fix the ultrasonic power at 180W, and maintain a constant temperature of 45℃ for 45 minutes.
[0067] Solid-liquid separation: After extraction, the material is removed and allowed to cool naturally to room temperature; centrifuge at 5000r / min for 10min and filter to remove solid residues of Gastrodia elata and collect the clear crude polysaccharide extract.
[0068] (4) Complete purification process of Gastrodia elata polysaccharide
[0069] First alcohol precipitation: Add 4 times the volume of anhydrous ethanol to the crude extract, add slowly while stirring, mix evenly, and then transfer to a 4°C refrigerated environment for alcohol precipitation for 12 hours.
[0070] Precipitation collection: Centrifuge at 5000 r / min for 10 min and collect the white polysaccharide precipitate from the bottom of the test tube;
[0071] Reconstitution and deproteinization: The polysaccharide precipitate was fully reconstituted in deionized water. Deproteinization reagent was added at a ratio of polysaccharide solution to Sevage reagent of 4:1 (V / V). Sevage reagent was prepared by mixing chloroform and n-butanol at a volume ratio of 4:1. After mixing, the mixture was vigorously shaken for 20 min, centrifuged at 7500 r / min for 25 min, and the layers were separated. The lower organic phase and the intermediate denatured protein layer were removed. The deproteinization process was repeated 3 to 5 times until there was no visible white flocculent protein precipitate at the interface between the two phases, and no characteristic protein absorption peak was detected at 280 nm by ultraviolet spectrophotometry.
[0072] Secondary alcohol precipitation dialysis: After deproteinization, the polysaccharide aqueous solution was added again with 4 times the volume of anhydrous ethanol and refrigerated at 4°C overnight for alcohol precipitation; after centrifugation to collect the precipitate, the polysaccharide precipitate was dissolved in deionized water, placed in a dialysis bag with a molecular weight cutoff of 3500 Da, and dialyzed with distilled water for 48 hours. The distilled water was changed every 8-12 hours during the dialysis process to completely remove DES small molecule impurities.
[0073] Drying and shaping: After dialysis, the polysaccharide aqueous solution is transferred to a freeze dryer and freeze-dried at low temperature to obtain pure white high-purity Gastrodia elata polysaccharide powder.
[0074] Example 2
[0075] A method for extracting polysaccharides from Gastrodia elata using an ultrasound-assisted eutectic solvent includes the following steps:
[0076] (1) Raw material pretreatment
[0077] Mature Gastrodia elata tubers from Zhaotong, Yunnan Province were selected and dried at a constant temperature of 45℃ in an electric heating forced-air drying oven until constant weight was achieved. The constant weight determination standard was that the difference in mass between two weighings of Gastrodia elata tubers at 2-hour intervals should not exceed 0.5 mg, and the moisture content of the dried Gastrodia elata raw material should be controlled below 5%. The dried Gastrodia elata tubers were crushed using a pulverizer and all were passed through a 40-mesh standard sieve. The sieved Gastrodia elata powder was sealed and stored at room temperature away from light for later use.
[0078] (2) Preparation of DES in eutectic solvent
[0079] Raw material ratio: betaine and glycerol, with a strictly controlled molar ratio of 1:2; add 25% deionized water by mass of the total solvent.
[0080] Synthesis procedure: Accurately weigh betaine and glycerol, add measured amount of deionized water and mix, place in an 80℃ constant temperature water bath, and continuously stir with magnetic force until the system forms a clear, transparent, homogeneous, non-layered, and non-crystallized viscous liquid.
[0081] Post-processing: Stop heating and allow to cool naturally to room temperature to obtain a special DES extraction solvent suitable for extracting polysaccharides from Gastrodia elata in Zhaotong.
[0082] (3) Ultrasound-assisted DES extraction of Gastrodia elata polysaccharide
[0083] Feeding ratio: Accurately weigh the pretreated Gastrodia elata powder, add the prepared DES solvent according to the material-liquid ratio of 1:35 (g:mL), and stir thoroughly to mix well;
[0084] Ultrasonic extraction parameters: Place the mixture in a probe-type ultrasonic processor, fix the ultrasonic power at 150W, and maintain a constant temperature of 50℃ for 35 minutes.
[0085] Solid-liquid separation: After extraction, the material is removed and allowed to cool naturally to room temperature; vacuum filtration is used to remove solid residues from Gastrodia elata and collect the clear crude polysaccharide extract.
[0086] (4) Complete purification process of Gastrodia elata polysaccharide
[0087] First alcohol precipitation: Add 4 times the volume of anhydrous ethanol to the crude extract, add slowly while stirring, mix evenly, and then transfer to a 4°C refrigerated environment for alcohol precipitation for 12 hours.
[0088] Precipitation collection: Centrifuge at 5000 r / min for 10 min and collect the white polysaccharide precipitate from the bottom of the test tube;
[0089] Reconstitution and deproteinization: The polysaccharide precipitate was fully reconstituted in deionized water. Deproteinization reagent was added at a ratio of polysaccharide solution to Sevage reagent of 4:1 (V / V). Sevage reagent was prepared by mixing chloroform and n-butanol at a volume ratio of 4:1. After mixing, the mixture was vigorously shaken for 20 min, centrifuged at 7500 r / min for 25 min, and the layers were separated. The lower organic phase and the intermediate denatured protein layer were removed. The deproteinization process was repeated 3 to 5 times until there was no visible white flocculent protein precipitate at the interface between the two phases, and no characteristic protein absorption peak was detected at 280 nm by ultraviolet spectrophotometry.
[0090] Secondary alcohol precipitation dialysis: After deproteinization, the polysaccharide aqueous solution was added again with 4 times the volume of anhydrous ethanol and refrigerated at 4°C overnight for alcohol precipitation; after centrifugation to collect the precipitate, the polysaccharide precipitate was dissolved in deionized water, placed in a dialysis bag with a molecular weight cutoff of 3500 Da, and dialyzed with distilled water for 48 hours. The distilled water was changed every 8-12 hours during the dialysis process to completely remove DES small molecule impurities.
[0091] Drying and shaping: After dialysis, the polysaccharide aqueous solution is transferred to a freeze dryer and freeze-dried at low temperature to obtain pure white high-purity Gastrodia elata polysaccharide powder.
[0092] Example 3
[0093] A method for extracting polysaccharides from Gastrodia elata using an ultrasound-assisted eutectic solvent includes the following steps:
[0094] (1) Raw material pretreatment
[0095] Mature Gastrodia elata tubers from Zhaotong, Yunnan Province were selected and dried at a constant temperature of 45℃ in an electric heating forced-air drying oven until constant weight was achieved. The constant weight determination standard was that the difference in mass between two weighings of Gastrodia elata tubers at 2-hour intervals should not exceed 0.5 mg, and the moisture content of the dried Gastrodia elata raw material should be controlled below 5%. The dried Gastrodia elata tubers were crushed using a pulverizer and all were passed through a 40-mesh standard sieve. The sieved Gastrodia elata powder was sealed and stored at room temperature away from light for later use.
[0096] (2) Preparation of DES in eutectic solvent
[0097] Raw material ratio: choline chloride and 1,2-propanediol, with a strictly controlled molar ratio of 2:1; add deionized water accounting for 35% of the total solvent mass;
[0098] Synthesis procedure: Accurately weigh choline chloride and 1,2-propanediol, add metered deionized water and mix, place in an 80°C constant temperature water bath, and continuously stir magnetically until the system forms a clear, transparent, homogeneous, non-layered, and non-crystallized viscous liquid.
[0099] Post-processing: Stop heating and allow to cool naturally to room temperature to obtain a special DES extraction solvent suitable for extracting polysaccharides from Gastrodia elata in Zhaotong.
[0100] (3) Ultrasound-assisted DES extraction of Gastrodia elata polysaccharide
[0101] Feeding ratio: Accurately weigh the pretreated Gastrodia elata powder, add the prepared DES solvent according to the material-liquid ratio of 1:30 (g:mL), and stir thoroughly to mix well;
[0102] Ultrasonic extraction parameters: Place the mixture in a probe-type ultrasonic processor, fix the ultrasonic power at 200W, and maintain a constant temperature of 45℃ for 35 minutes.
[0103] Solid-liquid separation: After extraction, the material is removed and allowed to cool naturally to room temperature; centrifuge at 5000r / min for 10min and filter to remove solid residues of Gastrodia elata and collect the clear crude polysaccharide extract.
[0104] (4) Complete purification process of Gastrodia elata polysaccharide
[0105] First alcohol precipitation: Add 4 times the volume of anhydrous ethanol to the crude extract, add slowly while stirring, mix evenly, and then transfer to a 4°C refrigerated environment for alcohol precipitation for 12 hours.
[0106] Precipitation collection: Centrifuge at 5000 r / min for 10 min and collect the white polysaccharide precipitate from the bottom of the test tube;
[0107] Reconstitution and deproteinization: The polysaccharide precipitate was fully reconstituted in deionized water. Deproteinization reagent was added at a ratio of polysaccharide solution to Sevage reagent of 4:1 (V / V). Sevage reagent was prepared by mixing chloroform and n-butanol at a volume ratio of 4:1. After mixing, the mixture was vigorously shaken for 20 min, centrifuged at 7500 r / min for 25 min, and the layers were separated. The lower organic phase and the intermediate denatured protein layer were removed. The deproteinization process was repeated 3 to 5 times until there was no visible white flocculent protein precipitate at the interface between the two phases, and no characteristic protein absorption peak was detected at 280 nm by ultraviolet spectrophotometry.
[0108] Secondary alcohol precipitation dialysis: After deproteinization, the polysaccharide aqueous solution was added again with 4 times the volume of anhydrous ethanol and refrigerated at 4°C overnight for alcohol precipitation; after centrifugation to collect the precipitate, the polysaccharide precipitate was dissolved in deionized water, placed in a dialysis bag with a molecular weight cutoff of 3500 Da, and dialyzed with distilled water for 48 hours. The distilled water was changed every 8-12 hours during the dialysis process to completely remove DES small molecule impurities.
[0109] Drying and shaping: After dialysis, the polysaccharide aqueous solution is transferred to a freeze dryer and freeze-dried at low temperature to obtain pure white high-purity Gastrodia elata polysaccharide powder.
[0110] Example 4
[0111] A method for extracting polysaccharides from Gastrodia elata using an ultrasound-assisted eutectic solvent includes the following steps:
[0112] (1) Raw material pretreatment
[0113] Mature Gastrodia elata tubers from Zhaotong, Yunnan Province were selected and dried at a constant temperature of 45℃ in an electric heating forced-air drying oven until constant weight was achieved. The constant weight determination standard was that the difference in mass between two weighings of Gastrodia elata tubers at 2-hour intervals should not exceed 0.5 mg, and the moisture content of the dried Gastrodia elata raw material should be controlled below 5%. The dried Gastrodia elata tubers were crushed using a pulverizer and all were passed through a 40-mesh standard sieve. The sieved Gastrodia elata powder was sealed and stored at room temperature away from light for later use.
[0114] (2) Preparation of DES in eutectic solvent
[0115] Raw material ratio: betaine and glucose, with a strictly controlled molar ratio of 1:1.5; add deionized water accounting for 45% of the total solvent mass;
[0116] Synthesis procedure: Accurately weigh betaine and glucose, add metered deionized water and mix, place in an 80℃ constant temperature water bath, and continuously stir with a magnetic force until the system forms a clear, transparent, homogeneous, non-layered, and non-crystallized viscous liquid.
[0117] Post-processing: Stop heating and allow to cool naturally to room temperature to obtain a special DES extraction solvent suitable for extracting polysaccharides from Gastrodia elata in Zhaotong.
[0118] (3) Ultrasound-assisted DES extraction of Gastrodia elata polysaccharide
[0119] Feeding ratio: Accurately weigh the pretreated Gastrodia elata powder, add the prepared DES solvent according to the material-liquid ratio of 1:45 (g:mL), and stir thoroughly to mix well;
[0120] Ultrasonic extraction parameters: Place the mixture in an industrial-grade ultrasonic extraction tank, fix the ultrasonic power at 150W, and maintain a constant temperature of 55℃ for 35 minutes.
[0121] Solid-liquid separation: After extraction, the material is removed and allowed to cool naturally to room temperature; centrifuge at 5000 r / min for 10 min or vacuum filter to remove solid residues of Gastrodia elata and collect the clear crude polysaccharide extract.
[0122] (4) Complete purification process of Gastrodia elata polysaccharide
[0123] First alcohol precipitation: Add 4 times the volume of anhydrous ethanol to the crude extract, add slowly while stirring, mix evenly, and then transfer to a 4°C refrigerated environment for alcohol precipitation for 12 hours.
[0124] Precipitation collection: Centrifuge at 5000 r / min for 10 min and collect the white polysaccharide precipitate from the bottom of the test tube;
[0125] Reconstitution and deproteinization: The polysaccharide precipitate was fully reconstituted in deionized water. Deproteinization reagent was added at a ratio of polysaccharide solution to Sevage reagent of 4:1 (V / V). Sevage reagent was prepared by mixing chloroform and n-butanol at a volume ratio of 4:1. After mixing, the mixture was vigorously shaken for 20 min, centrifuged at 7500 r / min for 25 min, and the layers were separated. The lower organic phase and the intermediate denatured protein layer were removed. The deproteinization process was repeated 3 to 5 times until there was no visible white flocculent protein precipitate at the interface between the two phases, and no characteristic protein absorption peak was detected at 280 nm by ultraviolet spectrophotometry.
[0126] Secondary alcohol precipitation dialysis: After deproteinization, the polysaccharide aqueous solution was added again with 4 times the volume of anhydrous ethanol and refrigerated at 4°C overnight for alcohol precipitation; after centrifugation to collect the precipitate, the polysaccharide precipitate was dissolved in deionized water, placed in a dialysis bag with a molecular weight cutoff of 3500 Da, and dialyzed with distilled water for 48 hours. The distilled water was changed every 8-12 hours during the dialysis process to completely remove DES small molecule impurities.
[0127] Drying and shaping: After dialysis, the polysaccharide aqueous solution is transferred to a freeze dryer and freeze-dried at low temperature to obtain pure white high-purity Gastrodia elata polysaccharide powder.
[0128] Example 5
[0129] A method for extracting polysaccharides from Gastrodia elata using an ultrasound-assisted eutectic solvent includes the following steps:
[0130] (1) Raw material pretreatment
[0131] Mature Gastrodia elata tubers from Zhaotong, Yunnan Province were selected and dried at a constant temperature of 45℃ in an electric heating forced-air drying oven until constant weight was achieved. The constant weight determination standard was that the difference in mass between two weighings of Gastrodia elata tubers at 2-hour intervals should not exceed 0.5 mg, and the moisture content of the dried Gastrodia elata raw material should be controlled below 5%. The dried Gastrodia elata tubers were crushed using a pulverizer and all were passed through a 40-mesh standard sieve. The sieved Gastrodia elata powder was sealed and stored at room temperature away from light for later use.
[0132] (2) Preparation of DES in eutectic solvent
[0133] Raw material ratio: choline chloride and glacial acetic acid, with a strictly controlled molar ratio of 2:1; add 20% deionized water by mass of the total solvent.
[0134] Synthesis procedure: Accurately weigh choline chloride and glacial acetic acid, add metered deionized water and mix, place in an 80℃ constant temperature water bath, and continuously stir with magnetic force until the system forms a clear, transparent, homogeneous, non-layered, and non-crystallized viscous liquid.
[0135] Post-processing: Stop heating and allow to cool naturally to room temperature to obtain a special DES extraction solvent suitable for extracting polysaccharides from Gastrodia elata in Zhaotong.
[0136] (3) Ultrasound-assisted DES extraction of Gastrodia elata polysaccharide
[0137] Feeding ratio: Accurately weigh the pretreated Gastrodia elata powder, add the prepared DES solvent according to the material-liquid ratio of 1:35 (g:mL), and stir thoroughly to mix well;
[0138] Ultrasonic extraction parameters: Place the mixture in a tank-type ultrasonic cleaner, fix the ultrasonic power at 250W, and maintain a constant temperature of 50℃ for 35 minutes.
[0139] Solid-liquid separation: After extraction, the material is removed and allowed to cool naturally to room temperature; centrifuge at 5000 r / min for 10 min to remove solid residue from Gastrodia elata and collect the clear crude polysaccharide extract.
[0140] (4) Complete purification process of Gastrodia elata polysaccharide
[0141] First alcohol precipitation: Add 4 times the volume of anhydrous ethanol to the crude extract, add slowly while stirring, mix evenly, and then transfer to a 4°C refrigerated environment for alcohol precipitation for 12 hours.
[0142] Precipitation collection: Centrifuge at 5000 r / min for 10 min and collect the white polysaccharide precipitate from the bottom of the test tube;
[0143] Reconstitution and deproteinization: The polysaccharide precipitate was fully reconstituted in deionized water. Deproteinization reagent was added at a ratio of polysaccharide solution to Sevage reagent of 4:1 (V / V). Sevage reagent was prepared by mixing chloroform and n-butanol at a volume ratio of 4:1. After mixing, the mixture was vigorously shaken for 20 min, centrifuged at 7500 r / min for 25 min, and the layers were separated. The lower organic phase and the intermediate denatured protein layer were removed. The deproteinization process was repeated 3 to 5 times until there was no visible white flocculent protein precipitate at the interface between the two phases, and no characteristic protein absorption peak was detected at 280 nm by ultraviolet spectrophotometry.
[0144] Secondary alcohol precipitation dialysis: After deproteinization, the polysaccharide aqueous solution was added again with 4 times the volume of anhydrous ethanol and refrigerated at 4°C overnight for alcohol precipitation; after centrifugation to collect the precipitate, the polysaccharide precipitate was dissolved in deionized water, placed in a dialysis bag with a molecular weight cutoff of 3500 Da, and dialyzed with distilled water for 48 hours. The distilled water was changed every 8-12 hours during the dialysis process to completely remove DES small molecule impurities.
[0145] Drying and shaping: After dialysis, the polysaccharide aqueous solution is transferred to a freeze dryer and freeze-dried at low temperature to obtain pure white high-purity Gastrodia elata polysaccharide powder.
[0146] Comparative Example 1
[0147] Water ultrasonic extraction of Gastrodia elata polysaccharides
[0148] Weigh 5 g of Gastrodia elata powder and add it to distilled water at a liquid-to-solid ratio of 20:1. Sonicate at 55℃ and 180 W for 45 min, cool to room temperature, and then add 4 times the original volume of anhydrous ethanol for alcohol precipitation. Transfer the mixture to a refrigerator at 4℃ and precipitate for 12 h. Centrifuge at 5000 r / min for 10 min. Collect the precipitate, add a specific volume of distilled water to redissolve it, and continue filtration to collect the Gastrodia elata polysaccharide aqueous solution.
[0149] Comparative Example 2
[0150] Enzymatic extraction of Gastrodia elata polysaccharides
[0151] For specific process details, please refer to Tan Sha, Zhu Renwei, Zhang Jing, et al. Optimization of enzymatic extraction process for Gastrodia elata polysaccharides [J]. Food Research and Development, 2017, 38(15): 50-53.
[0152] Experimental Example 1
[0153] Validation method for quantitative detection of polysaccharides (phenol-sulfuric acid method)
[0154] This method, used to verify the extraction effect of polysaccharides in Example 1, is not part of the extraction process, but rather a standard method for quantifying and verifying the process effect.
[0155] Reagent preparation: Prepare a 5% (w / w) aqueous solution of phenol, and prepare a gradient standard solution using D-glucose as the standard;
[0156] Colorimetric detection: Take 1 mL of the polysaccharide solution to be tested, add 1 mL of 5% phenol solution and 5 mL of concentrated sulfuric acid in sequence, shake to mix, and then incubate at 45℃ for 25 min for color development. Cool to room temperature. Use pure water as a blank control group. Measure the absorbance at 490 nm wavelength with a UV spectrophotometer. Measure each sample in parallel 3 times.
[0157] Standard curve: A standard curve was plotted with the glucose standard concentration C (mg / mL) on the ordinate and the measured absorbance A at 490 nm on the abscissa. Linear regression was then performed, yielding the regression equation: C = 0.0969A + 0.0003, R0 2 =0.9993 where C is the polysaccharide concentration (mg / mL) and A is the measured absorbance value at a wavelength of 490 nm;
[0158] The formula for calculating the yield is: Gastrodia elata polysaccharide yield (%) = (C × V × N / m) × 100% Where: C is the polysaccharide concentration of the test solution (mg / mL); V is the volume of the test solution (mL); N is the dilution factor of the solution; and m is the mass of Gastrodia elata powder raw material (g).
[0159] Experimental Example 2
[0160] Effects of different DES on the yield of Gastrodia elata polysaccharides
[0161] Different DES settings are configured, specifically including the following 21 groups:
[0162] DES1: Choline chloride:DL-malic acid = 1:1;
[0163] DES2: Choline chloride:DL-malic acid = 1:2;
[0164] DES3: Choline chloride:DL-malic acid = 2:1;
[0165] DES4: Choline chloride: Glacial acetic acid = 1:1;
[0166] DES5: Choline chloride:Glacial acetic acid = 1:2;
[0167] DES6: Choline chloride:Glacial acetic acid = 2:1;
[0168] DES7: Choline chloride: Oxalic acid = 1:1;
[0169] DES8: Choline chloride: Oxalic acid = 1:2;
[0170] DES9: Choline chloride: Oxalic acid = 2:1;
[0171] DES10: Choline chloride:glucose = 1:1;
[0172] DES11: Choline chloride:glucose = 1:2;
[0173] DES12: Choline chloride:glucose = 2:1;
[0174] DES13: Choline chloride: 1,2-propanediol = 1:1;
[0175] DES14: Choline chloride: 1,2-propanediol = 1:2;
[0176] DES15: Choline chloride: 1,2-propanediol = 2:1;
[0177] DES16: Choline chloride: Isopropanol = 1:1;
[0178] DES17: Choline chloride:Isopropanol = 1:2;
[0179] DES18: Choline chloride:Isopropanol = 2:1;
[0180] DES19: Choline chloride: Ethylene glycol = 1:1;
[0181] DES20: Choline chloride: Ethylene glycol = 1:2;
[0182] DES21: Choline chloride: Ethylene glycol = 2:1.
[0183] from Figure 1 The results show that among the 21 DES formulations screened, the choline chloride and DL-malic acid (1:1) system exhibited the best extraction effect for Gastrodia elata polysaccharides, with an average yield of 19.75%, significantly higher than the pure water control group and other DES combinations. This is because the DES formed by this ratio achieves synergistic optimization of hydrogen bonding, acidity, and viscosity: choline chloride, as a hydrogen bond acceptor, forms a stable hydrogen bond network with DL-malic acid (a polycarboxyl / hydroxyl donor), which can efficiently disrupt the cell wall of Gastrodia elata and promote polysaccharide dissolution; at the same time, the system is mildly acidic, avoiding polysaccharide degradation caused by a strong acid environment and preventing polysaccharide precipitation due to excessive ionic strength, and the moderate viscosity ensures good tissue penetration and mass transfer efficiency, thus resulting in the best extraction effect.
[0184] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention, including but not limited to:
[0185] 1. Replacement of deproteinization process: Sevage reagent deproteinization can be replaced by trichloroacetic acid (TCA) method and protease deproteinization. Only the final purity of polysaccharide is slightly reduced, which does not affect the overall extraction yield.
[0186] 2. Drying process replacement: Freeze drying can replace vacuum low-temperature drying and spray drying (with the addition of excipients such as maltodextrin), both of which can yield solid Gastrodia elata polysaccharide products;
[0187] 3. Solid-liquid separation replacement: Centrifugal separation can replace industrial solid-liquid separation equipment such as vacuum filtration and plate and frame filter press, and is suitable for different scale production scenarios.
[0188] 4. Raw material alternatives: The extraction method and DES composition of the present invention can also be applied to the polysaccharide extraction of other Gastrodia elata variants (such as red Gastrodia elata and green Gastrodia elata) or other medicinal and edible Chinese medicinal materials rich in polysaccharides (such as Astragalus membranaceus, Ganoderma lucidum and Poria cocos), but Zhaotong black Gastrodia elata is the most preferred raw material.
[0189] 5. Alternatives to ultrasonic equipment: The ultrasonic-assisted extraction described in this invention is not limited to a tank-type ultrasonic cleaner. Probe-type ultrasonic processors, continuous flow ultrasonic extraction equipment, or industrial ultrasonic extraction tanks can also be used, as long as the power density (W / mL) is adjusted to an equivalent level to that of this invention.
[0190] In summary, in accordance with the principles of this invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.
Claims
1. A method for extracting polysaccharides from Gastrodia elata using an ultrasound-assisted eutectic solvent, comprising the following steps: (1) Raw material pretreatment: Select mature black Gastrodia tuber from Zhaotong, Yunnan, dry at 45℃ to constant weight, and pulverize to 40 mesh to obtain Gastrodia powder for later use; (2) Preparation of DES extraction solvent: Take quaternary ammonium salt hydrogen bond acceptor and oxygen-containing organic complexing agent hydrogen bond donor with a molar ratio of 1:2 to 2:1, add 20% to 50% of deionized water in total solvent mass, and continuously stir magnetically in a constant temperature water bath at 80℃ until the liquid is homogeneous. Stop heating and let it cool naturally to room temperature to obtain DES extraction solvent. (3) Ultrasonic-assisted DES extraction of crude extract of Gastrodia elata polysaccharide: Gastrodia elata powder was added to DES solvent at a material-liquid ratio of 1g:30~50mL, stirred and mixed thoroughly, and ultrasonic extraction was performed at ultrasonic power of 100~300W and 35~55℃ for 35~55min. After extraction, the material was removed and allowed to cool naturally to room temperature. After filtration, the clear crude extract of polysaccharide was collected. (4) Purification of Gastrodia elata polysaccharide: Add 4 times the volume of anhydrous ethanol to the crude extract, add slowly while stirring, mix evenly, transfer to a 4℃ cold storage environment and let it stand for ethanol precipitation for 12 hours; then centrifuge and collect the polysaccharide precipitate. (5) First alcohol precipitation: Add deionized water to the polysaccharide precipitate to fully reconstitute it and obtain a polysaccharide solution. Add deproteinizing reagent after mixing the polysaccharide solution with Sevage reagent. After mixing, shake vigorously for 20 min, centrifuge at 7500 r / min for 25 min, and remove the lower organic phase and the intermediate protein denaturation layer after separation. Repeat the deproteinization operation 3 to 5 times until there is no visible white flocculent protein precipitate at the interface between the two phases, and no protein characteristic absorption peak is detected at 280 nm by ultraviolet spectrophotometry. (6) Secondary alcohol precipitation dialysis: After deproteinization, the polysaccharide aqueous solution was added again with 4 times the volume of anhydrous ethanol and refrigerated at 4°C overnight for alcohol precipitation; after centrifugation to collect the precipitate, the polysaccharide precipitate was dissolved in deionized water, put into a dialysis bag, and DES small molecule impurities were completely removed. (7) Drying and shaping: After dialysis, the polysaccharide aqueous solution is transferred to a freeze dryer and freeze-dried at low temperature to obtain Gastrodia elata polysaccharide powder.
2. The method according to claim 1, wherein: The moisture content of the dried Gastrodia elata raw material in step (1) is <5%.
3. The method according to claim 1, wherein: The quaternary ammonium salt hydrogen bond acceptors mentioned in step (2) include: choline chloride and betaine; The oxygen-containing organic complexing agents include: DL-malic acid, citric acid, lactic acid, ethylene glycol, 1,2-propanediol, glycerol, glucose, fructose, glacial acetic acid, and oxalic acid.
4. The method according to claim 3, wherein: The quaternary ammonium salt hydrogen bond acceptor is choline chloride; The oxygen-containing organic complexing agent is DL-malic acid.
5. The method according to claim 1, wherein: The ratio of the nutrient solution in step (3) is 1g:50mL; The ultrasonic power was 180W, and the extraction was carried out continuously at 45℃ for 45 minutes.
6. The method according to claim 1, wherein: In step (4), the centrifugation speed is 5000 r / min and the centrifugation time is 10 min.
7. The method according to claim 1, wherein: The volume ratio of the polysaccharide solution to Sevage reagent in step (5) is 4:1; The Sevage reagent is prepared by mixing chloroform and n-butanol in a volume ratio of 4:
1.
8. The method according to claim 1, wherein: The dialysis bag mentioned in step (6) is a dialysis bag with a molecular weight cutoff of 3500 Da; The dialysis was performed using distilled water, with a total dialysis time of 48 hours, during which the distilled water was changed every 8 to 12 hours.
9. A polysaccharide from Gastrodia elata prepared by any one of the methods described in claims 1 to 8.
10. The application of the Gastrodia elata polysaccharide according to claim 9 in the preparation of functional foods or health foods.