Method for extracting lobetyolin

Abstract

The invention relates to the technical field of extraction of effective components of traditional Chinese medicines, in particular to an extraction method of lobetyolin. The extraction method comprises the following steps: mixing codonopsis pilosula, a compound enzyme and a buffer solution, and carrying out enzymolysis to obtain an enzymatic hydrolysate; mixing the enzymatic hydrolysate with an ethanol solution, carrying out ultrasonic-microwave synergistic extraction, and collecting supernate after the extraction is finished; treating the supernate through a micro-filtration membrane, an ultra-filtration membrane and a nano-filtration membrane in sequence, and collecting a nano-filtration trapped fluid; and concentrating the nanofiltration trapped fluid under reduced pressure, and drying to obtain the lobetyolin extract. By combining composite enzymolysis, ultrasonic-microwave synergistic extraction and three-stage membrane filtration, the invention provides the lobetyolin extraction method which is mild in condition, high in extraction efficiency, stable in process and environment-friendly.

Description

Technical Field

[0001] This invention relates to the field of extraction technology of effective components of traditional Chinese medicine, and in particular to a method for extracting codonopsis glycosides. Background Technology

[0002] Codonopsis pilosula glycosides are one of the main active components of Codonopsis pilosula, possessing various pharmacological activities such as anti-ulcer, antioxidant, anti-inflammatory, and gastric mucosal protection. Existing methods for extracting codonopsis pilosula glycosides include water decoction, alcohol reflux, and ultrasonic extraction. However, these methods generally suffer from low extraction efficiency, large amounts of organic solvents, and complex processes. For example, traditional water decoction involves high temperatures and long extraction times, easily leading to structural damage to codonopsis pilosula glycosides; alcohol reflux uses large amounts of organic solvents, posing safety risks and requiring complex subsequent processing; while ultrasonic extraction has a short extraction time, its extraction efficiency is unstable. Therefore, developing a mild, efficient, stable, and simple method for extracting codonopsis pilosula glycosides is of great significance. Summary of the Invention

[0003] The purpose of this invention is to address the problems of low extraction efficiency, large organic solvent consumption, and complex processes in existing methods for extracting codonopsis glycosides. This invention provides a method for extracting codonopsis glycosides by combining compound enzymatic hydrolysis, ultrasonic-microwave synergistic extraction, and three-stage membrane filtration. This method offers a mild, efficient, stable, and environmentally friendly extraction method for codonopsis glycosides.

[0004] To achieve the above objectives, the present invention provides a method for extracting codonopsis glycosides, comprising the following steps: (1) Mix Codonopsis pilosula, compound enzyme and buffer solution, and perform enzymatic hydrolysis to obtain enzymatic hydrolysate; (2) Mix the enzyme hydrolysate and ethanol solution and perform ultrasonic-microwave synergistic extraction. After extraction, collect the supernatant. (3) The supernatant is sequentially passed through a microfiltration membrane, an ultrafiltration membrane, and a nanofiltration membrane, and the nanofiltration retentate is collected; (4) The nanofiltration retentate was concentrated under reduced pressure and then dried to obtain the Codonopsis pilosula glycoside extract.

[0005] Preferably, in step (1), the complex enzyme includes cellulase, pectinase and amylase; the mass ratio of cellulase, pectinase and amylase is (1-3):(1-2):(0.5-1).

[0006] Preferably, in step (1), the buffer solution is disodium hydrogen phosphate-citric acid buffer solution; the pH of the buffer solution is 4.5-5.5.

[0007] Preferably, in step (1), the mass ratio of Codonopsis pilosula to the compound enzyme is 100:(0.2-0.5); the mass-volume ratio of Codonopsis pilosula to the buffer solution is 1g:(10-15)mL.

[0008] Preferably, in step (1), the enzymatic hydrolysis temperature is 45-55℃ and the time is 2-4h.

[0009] Preferably, in step (2), the volume fraction of ethanol in the mixed solution is 50-70%; the material-to-liquid ratio of the mixed solution is 1g:(15-25)mL.

[0010] Preferably, in step (2), the ultrasonic power of the ultrasonic-microwave synergistic extraction is 300-500W, the microwave power is 400-600W, the temperature is 40-50℃, and the extraction time is 10-20min.

[0011] Preferably, in step (3), the pore size of the microfiltration membrane is 0.05-0.2 micrometers; the molecular weight cutoff of the ultrafiltration membrane is 50,000-100,000 Daltons; and the molecular weight cutoff of the nanofiltration membrane is 200-500 Daltons.

[0012] Preferably, in step (4), the vacuum concentration is carried out at 40-50°C, and the vacuum concentration is carried out until there is no alcohol odor, and then the product is dried.

[0013] Preferably, in step (4), the drying is vacuum drying, the drying temperature is 40-50℃, and the drying time is 4-6h.

[0014] The beneficial effects of this invention are as follows: 1. This invention provides a method for extracting codonopsis glycosides, comprising the following steps: mixing Codonopsis pilosula, a compound enzyme, and a buffer solution for enzymatic hydrolysis to obtain an enzymatic hydrolysate; mixing the enzymatic hydrolysate with an ethanol solution for ultrasonic-microwave synergistic extraction; collecting the supernatant after extraction; sequentially passing the supernatant through a microfiltration membrane, an ultrafiltration membrane, and a nanofiltration membrane, collecting the nanofiltration retentate; concentrating the nanofiltration retentate under reduced pressure and then drying it to obtain the codonopsis glycoside extract. This invention provides a mild, efficient, stable, and environmentally friendly method for extracting codonopsis glycosides by combining compound enzymatic hydrolysis, ultrasonic-microwave synergistic extraction, and three-stage membrane filtration.

[0015] 2. This invention utilizes a complex enzyme to efficiently enzymatically hydrolyze the cell wall of *Codonopsis pilosula* under mild conditions, effectively disrupting its structure and promoting the full release of codonopsis glycosides. Building upon this, it combines ultrasonic-microwave synergistic extraction technology, leveraging the synergistic effect of cavitation and internal heating to significantly improve the dissolution rate and extraction efficiency of the target components. Furthermore, a three-stage membrane filtration system (microfiltration-ultrafiltration-nanofiltration) replaces the complex purification steps of traditional multiple extractions and precipitation, simplifying the operation process, shortening the production cycle, and effectively removing impurities such as large-molecule proteins, polysaccharides, and some small-molecule inorganic salts, achieving precise retention and purification of codonopsis glycosides. The final extract obtained by this integrated process has a high content of codonopsis glycosides, good process reproducibility, stable product quality across batches, and an environmentally friendly overall process, making it easy to scale up for application.

[0016] 3. The temperature of the entire extraction process of this invention is always controlled below 55℃, which effectively avoids the decomposition or structural changes of Codonopsis pilosula glycosides caused by high temperature, and ensures the activity and quality stability of the product.

[0017] 4. In addition, enzymatic pretreatment effectively improves the dissolution efficiency of codonopsis glycosides in the raw materials, thereby reducing the amount of ethanol solution used in the subsequent extraction process; on this basis, ultrasonic-microwave assisted extraction technology further reduces the total solvent demand and shortens the extraction time; in addition, the application of nanofiltration membrane can realize online recovery and simultaneous concentration of ethanol solvent, which significantly reduces the amount of organic solvent used and emitted, making the entire extraction process more in line with green and environmentally friendly requirements. Detailed Implementation

[0018] This invention provides a method for extracting codonopsis glycosides, comprising the following steps: (1) Mix Codonopsis pilosula, compound enzyme and buffer solution, and perform enzymatic hydrolysis to obtain enzymatic hydrolysate; (2) Mix the enzyme hydrolysate and ethanol solution and perform ultrasonic-microwave synergistic extraction. After extraction, collect the supernatant. (3) The supernatant is sequentially passed through a microfiltration membrane, an ultrafiltration membrane, and a nanofiltration membrane, and the nanofiltration retentate is collected; (4) The nanofiltration retentate was concentrated under reduced pressure and then dried to obtain the Codonopsis pilosula glycoside extract.

[0019] In this invention, in step (1), Codonopsis pilosula is pretreated before use. The pretreatment includes drying, pulverizing, and passing Codonopsis pilosula through a 40-80 mesh sieve.

[0020] In this invention, in step (1), the complex enzyme includes cellulase, pectinase and amylase; the mass ratio of cellulase, pectinase and amylase is (1-3):(1-2):(0.5-1).

[0021] Cellulase, pectinase, and amylase are all food-grade or pharmaceutical-grade industrial enzyme preparations, which are commercially available (e.g., products provided by manufacturers such as Nanning Dongheng Huadao Biotechnology Co., Ltd. and Jiangsu Ruiyang Biotechnology Co., Ltd.).

[0022] Cellulase has an enzyme activity of ≥10000 U / g. The activity unit is defined by the filter paper method (FPA method), which is the amount of enzyme required to produce 1 micromole of glucose (or reducing sugar in glucose form) per minute from the filter paper substrate under specific conditions. Pectinase has an enzyme activity of ≥10000 U / g, which is defined as the amount of enzyme required to catalyze the production of 1 micromole of galacturonic acid from pectin per minute under specific conditions. Amylase has an enzyme activity of ≥10000 U / g, which is defined as the amount of enzyme required to liquefy 1 g of soluble starch per hour under specific conditions.

[0023] In this invention, in step (1), the buffer solution is disodium hydrogen phosphate-citric acid buffer solution; the pH of the buffer solution is 4.5-5.5.

[0024] This buffer solution can be prepared using conventional methods in the art, for example, by preparing separate 0.1 mol / L citric acid solutions and 0.2 mol / L disodium hydrogen phosphate solutions, then mixing them at a specific volume ratio, and calibrating to the desired pH value using a precision pH meter. Alternatively, commercially available buffer kits or ready-made buffer solutions with the corresponding pH values ​​can be used directly.

[0025] In this invention, in step (1), the mass ratio of Codonopsis pilosula to the compound enzyme is 100:(0.2-0.5); the mass-volume ratio of Codonopsis pilosula to the buffer solution is 1g:(10-15)mL.

[0026] In this invention, in step (1), the enzymatic hydrolysis temperature is 45-55℃ and the time is 2-4h.

[0027] In this invention, in step (2), the volume fraction of ethanol in the mixed solution is 50-70%; the material-to-liquid ratio of the mixed solution is 1g:(15-25)mL.

[0028] The material-liquid ratio refers to the proportional relationship between the mass of solid raw materials and the volume of liquid solvent in the mixed solution.

[0029] In this invention, in step (2), the ultrasonic power of the ultrasonic-microwave synergistic extraction is 300-500W, the microwave power is 400-600W, the temperature is 40-50℃, and the extraction time is 10-20min.

[0030] In this invention, in step (2), after extraction, solid-liquid separation is performed and the supernatant is collected; the solid-liquid separation is performed by centrifugation at 8000-10000 r / min for 15-20 min.

[0031] In this invention, in step (3), the pore size of the microfiltration membrane is 0.05-0.2 micrometers; the molecular weight cutoff of the ultrafiltration membrane is 50,000-100,000 Daltons; and the molecular weight cutoff of the nanofiltration membrane is 200-500 Daltons.

[0032] In this invention, in step (4), the vacuum concentration is carried out at 40-50°C, and the vacuum concentration is carried out until there is no alcohol smell (the vacuum concentration is carried out until there is no obvious ethanol smell) and then the product is dried.

[0033] In this invention, in step (4), the drying is vacuum drying, the drying temperature is 40-50℃, and the drying time is 4-6h.

[0034] The present invention will be further described below with reference to embodiments. Unless otherwise defined, the technical or scientific terms used in this invention should have the ordinary meaning understood by one of ordinary skill in the art. The features mentioned above or in the specific examples mentioned in this invention can be combined arbitrarily, and these specific embodiments are only used to illustrate the invention and are not intended to limit the scope of the invention.

[0035] Example 1 This embodiment provides a method for extracting codonopsis glycosides, including the following steps: The Codonopsis pilosula was dried, pulverized, and passed through a 50-mesh sieve to obtain Codonopsis pilosula powder.

[0036] A complex enzyme (cellulase, pectinase, and amylase in a mass ratio of 2:1:1) and a disodium hydrogen phosphate-citric acid buffer solution with a pH of 5.0 were added sequentially to the Codonopsis pilosula powder. The mass ratio of Codonopsis pilosula to the complex enzyme was 100:0.3, and the mass-volume ratio of Codonopsis pilosula to the buffer solution was 1g:12mL. The mixture was enzymatically hydrolyzed at 50℃ for 3 hours to obtain the enzymatic hydrolysate.

[0037] Add ethanol solution to the enzymatic hydrolysate to adjust the volume fraction of ethanol in the mixed solution to 60%, and the material-to-liquid ratio to 1g:20mL; perform ultrasonic-microwave synergistic extraction, setting the ultrasonic power to 400W, the microwave power to 500W, the extraction temperature to 45℃, and the extraction time to 15min; after extraction, centrifuge at 9000r / min for 18min and collect the supernatant.

[0038] The supernatant was sequentially passed through a microfiltration membrane (pore size of 0.1 micrometers), an ultrafiltration membrane (molecular weight cutoff of 50,000 Daltons), and a nanofiltration membrane (molecular weight cutoff of 300 Daltons), and the nanofiltration retentate was collected.

[0039] The nanofiltration retentate was concentrated under reduced pressure at 45°C until no alcohol odor was detected, and then vacuum dried (at 50°C for 5 hours) to obtain the Codonopsis pilosula glycoside extract.

[0040] Example 2 This embodiment provides a method for extracting codonopsis glycosides, including the following steps: The Codonopsis pilosula was dried, pulverized, and passed through a 60-mesh sieve to obtain Codonopsis pilosula powder.

[0041] A complex enzyme (cellulase, pectinase, and amylase in a mass ratio of 1.5:1:0.5) and a disodium hydrogen phosphate-citric acid buffer solution with a pH of 4.8 were added sequentially to the Codonopsis pilosula powder. The mass ratio of Codonopsis pilosula to the complex enzyme was 100:0.4, and the mass-volume ratio of Codonopsis pilosula to the buffer solution was 1g:10mL. The mixture was enzymatically hydrolyzed at 50℃ for 3 hours to obtain the enzymatic hydrolysate.

[0042] Add ethanol solution to the enzymatic hydrolysate to adjust the volume fraction of ethanol in the mixed solution to 55%, and the material-to-liquid ratio to 1g:18mL; perform ultrasonic-microwave synergistic extraction, setting the ultrasonic power to 350W, the microwave power to 450W, the extraction temperature to 48℃, and the extraction time to 18min; after extraction, centrifuge at 10000r / min for 15min and collect the supernatant.

[0043] The supernatant was sequentially passed through a microfiltration membrane (pore size of 0.05 micrometers), an ultrafiltration membrane (molecular weight cutoff of 80,000 Daltons), and a nanofiltration membrane (molecular weight cutoff of 400 Daltons), and the nanofiltration retentate was collected.

[0044] The nanofiltration retentate was concentrated under reduced pressure at 50°C until no alcohol odor remained, and then vacuum dried (at 45°C for 5 hours) to obtain the Codonopsis pilosula glycoside extract.

[0045] Example 3 This embodiment provides a method for extracting codonopsis glycosides, including the following steps: The Codonopsis pilosula was dried, pulverized, and passed through an 80-mesh sieve to obtain Codonopsis pilosula powder.

[0046] A complex enzyme (cellulase, pectinase, and amylase in a mass ratio of 3:2:1) and a disodium hydrogen phosphate-citric acid buffer solution with a pH of 5.2 were added sequentially to the Codonopsis pilosula powder. The mass ratio of Codonopsis pilosula to the complex enzyme was 100:0.3, and the mass-volume ratio of Codonopsis pilosula to the buffer solution was 1g:15mL. The mixture was enzymatically hydrolyzed at 55℃ for 2 hours to obtain the enzymatic hydrolysate.

[0047] Add ethanol solution to the enzymatic hydrolysate to adjust the volume fraction of ethanol in the mixed solution to 65%, and the material-to-liquid ratio to 1g:22mL; perform ultrasonic-microwave synergistic extraction, setting the ultrasonic power to 500W, the microwave power to 600W, the extraction temperature to 50℃, and the extraction time to 12min; after extraction, centrifuge at 8000r / min for 20min and collect the supernatant.

[0048] The supernatant was sequentially passed through a microfiltration membrane (pore size of 0.2 micrometers), an ultrafiltration membrane (molecular weight cutoff of 100,000 Daltons), and a nanofiltration membrane (molecular weight cutoff of 200 Daltons), and the nanofiltration retentate was collected.

[0049] The nanofiltration retentate was concentrated under reduced pressure at 40°C until no alcohol odor was detected, and then vacuum dried (at 45°C for 6 hours) to obtain the Codonopsis pilosula glycoside extract.

[0050] Comparative Example 1 This comparative example provides a method for extracting codonopsis glycosides, including the following steps: The Codonopsis pilosula was dried, pulverized, and passed through a 60-mesh sieve to obtain Codonopsis pilosula powder.

[0051] Take 100g of Codonopsis pilosula powder, place it in a round-bottom flask, add 1800mL of 60% ethanol solution, and reflux at 85℃ for 2h. Filter the extract. Add another 1800mL of 60% ethanol solution to the residue, and reflux at 85℃ for another 2h. Combine the two extracts. Concentrate the combined extract under reduced pressure at 60℃ until it becomes a paste (without obvious fluidity). Then transfer the paste to a vacuum drying oven and dry at 50℃ for 5h to obtain Codonopsis pilosula glycoside extract.

[0052] Comparative Example 2 This comparative example provides a method for extracting codonopsis glycosides, which differs from Example 1 in that the extraction temperature is modified to 65°C.

[0053] Comparative Example 3 This comparative example provides a method for extracting codonopsis glycosides, which differs from Example 1 in that the addition of the complex enzyme is omitted, and specifically includes the following steps: The Codonopsis pilosula was dried, pulverized, and passed through a 50-mesh sieve to obtain Codonopsis pilosula powder.

[0054] Add disodium hydrogen phosphate-citric acid buffer solution (pH 5.0) to Codonopsis pilosula powder (mass-volume ratio of Codonopsis pilosula to buffer solution is 1 g: 12 mL), mix well, and let stand at 50℃ for 3 h to obtain a mixed solution.

[0055] Add ethanol solution to the mixture to adjust the volume fraction of ethanol in the mixture to 60% and the material-liquid ratio to 1g:20mL; perform ultrasonic-microwave synergistic extraction, setting the ultrasonic power to 400W, the microwave power to 500W, the extraction temperature to 45℃, and the extraction time to 15min; after extraction, centrifuge at 9000r / min for 18min and collect the supernatant.

[0056] The supernatant was sequentially passed through a microfiltration membrane (pore size of 0.1 micrometers), an ultrafiltration membrane (molecular weight cutoff of 50,000 Daltons), and a nanofiltration membrane (molecular weight cutoff of 300 Daltons), and the nanofiltration retentate was collected.

[0057] The nanofiltration retentate was concentrated under reduced pressure at 45°C until no alcohol odor was detected, and then vacuum dried (at 50°C for 5 hours) to obtain the Codonopsis pilosula glycoside extract.

[0058] Experimental Example 1 The content and extraction rate of codonopsis glycosides in the extracts of codonopsis glycosides obtained in Examples 1-3 and Comparative Examples 1-3 were determined by high performance liquid chromatography. The specific methods are as follows: (1) Chromatographic conditions: The chromatographic column was a C18 reversed-phase column (column length 250 mm, inner diameter 4.6 mm, particle size of packing particles 5 μm), the mobile phase was a mixed solution of acetonitrile and water (volume ratio of acetonitrile and water 25:75), the flow rate was 1.0 mL / min, the column temperature was 30℃, the detection wavelength was 268 nm, and the injection volume was 10 μL. (2) Preparation of standard curve: An appropriate amount of codonopsis glycoside reference standard was accurately weighed, dissolved and diluted with methanol to prepare a series of standard solutions with concentrations (5, 10, 20, 40, 80 μg / mL), and injected for determination under the above chromatographic conditions. The standard curve equation Y = aX + b(r) was obtained by linear regression of peak area (Y) against concentration (X, μg / mL). 2 (Greater than 0.999), indicating that the linear relationship of codonopsis glycosides is good in the range of 5-80 μg / mL. (3) Sample processing and determination: Accurately weigh an appropriate amount of codonopsis glycoside extract obtained from each example and comparative example, dissolve and dilute with methanol, filter through a 0.45 μm microporous membrane, and analyze by injection under the above chromatographic conditions. Record the peak area of ​​codonopsis glycosides and substitute it into the standard curve to calculate its content. (4) Calculation of content and extraction rate: Codonopsis glycoside content (%) = (mass of codonopsis glycosides in the extract / mass of codonopsis glycoside extract) × 100%; Codonopsis glycoside extraction rate (%) = (mass of codonopsis glycosides in the extract / mass of raw codonopsis) × 100%. The obtained determination results are recorded in Table 1.

[0059] Table 1 Measurement Results

[0060] Table 1 shows that the content and extraction rate of codonopsis glycosides in the extracts prepared in Examples 1-3 of this invention are significantly higher than those in Comparative Examples 1-3. The results indicate that the extraction method provided by this invention achieves efficient and stable extraction of codonopsis glycosides under mild conditions, and has advantages such as high extraction efficiency, simple process, and low solvent consumption.

[0061] Therefore, this invention provides a mild, efficient, stable, and environmentally friendly method for extracting codonopsis glycosides by combining enzymatic hydrolysis, ultrasonic-microwave synergistic extraction, and three-stage membrane filtration.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for extracting codonopsis glycosides, characterized in that, Includes the following steps: (1) Mix Codonopsis pilosula, compound enzyme and buffer solution, and perform enzymatic hydrolysis to obtain enzymatic hydrolysate; (2) Mix the enzyme hydrolysate and ethanol solution and perform ultrasonic-microwave synergistic extraction. After extraction, collect the supernatant. (3) The supernatant is sequentially passed through a microfiltration membrane, an ultrafiltration membrane, and a nanofiltration membrane, and the nanofiltration retentate is collected; (4) The nanofiltration retentate was concentrated under reduced pressure and then dried to obtain the Codonopsis pilosula glycoside extract.

2. The method for extracting codonopsis glycosides according to claim 1, characterized in that, In step (1), the complex enzyme includes cellulase, pectinase and amylase; the mass ratio of cellulase, pectinase and amylase is (1-3):(1-2):(0.5-1).

3. The method for extracting codonopsis glycosides according to claim 1, characterized in that, In step (1), the buffer solution is disodium hydrogen phosphate-citric acid buffer solution; the pH of the buffer solution is 4.5-5.

5.

4. The method for extracting codonopsis glycosides according to claim 1, characterized in that, In step (1), the mass ratio of Codonopsis pilosula to the compound enzyme is 100:(0.2-0.5); the mass-volume ratio of Codonopsis pilosula to the buffer solution is 1g:(10-15)mL.

5. The method for extracting codonopsis glycosides according to claim 1, characterized in that, In step (1), the enzymatic hydrolysis temperature is 45-55℃ and the time is 2-4h.

6. The method for extracting codonopsis glycosides according to claim 1, characterized in that, In step (2), the volume fraction of ethanol in the mixed solution is 50-70%; the material-to-liquid ratio of the mixed solution is 1g:(15-25)mL.

7. The method for extracting codonopsis glycosides according to claim 1, characterized in that, In step (2), the ultrasonic power of the ultrasonic-microwave synergistic extraction is 300-500W, the microwave power is 400-600W, the temperature is 40-50℃, and the extraction time is 10-20min.

8. The method for extracting codonopsis glycosides according to claim 1, characterized in that, In step (3), the pore size of the microfiltration membrane is 0.05-0.2 micrometers; the molecular weight cutoff of the ultrafiltration membrane is 50,000-100,000 Daltons; and the molecular weight cutoff of the nanofiltration membrane is 200-500 Daltons.

9. The method for extracting codonopsis glycosides according to claim 1, characterized in that, In step (4), the vacuum concentration is carried out at 40-50℃, and after vacuum concentration until there is no alcohol odor, it is dried.

10. The method for extracting codonopsis glycosides according to claim 1, characterized in that, In step (4), the drying is vacuum drying, the drying temperature is 40-50℃, and the time is 4-6h.

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