Preparation method of astragalus polysaccharide extract

Abstract

The invention belongs to the technical field of extraction of traditional Chinese medicinal materials, and relates to a preparation method of an astragalus polysaccharide extract. The method comprises the following steps: adding astragalus membranaceus powder into a deep-eutectic solvent water solution containing cellulase, carrying out ultrasonic extraction at 25-75 DEG C for 15-60 minutes, centrifuging, taking supernatant and carrying out alcohol precipitation to obtain astragalus membranaceus polysaccharide; the eutectic solvent in the eutectic solvent aqueous solution comprises one of choline chloride and glycerol, choline chloride and lactic acid, and choline chloride and urea. The astragalus polysaccharide can be efficiently extracted by adopting the method, and the method is obviously higher than a traditional water extraction method. According to the method, a natural deep-eutectic solvent method, an enzymatic extraction method and an ultrasonic-assisted extraction method are fused into an enzyme-assisted natural deep-eutectic solvent-ultrasonic-assisted extraction method for extracting the polysaccharide from the traditional Chinese medicinal material astragalus membranaceus, the extraction rate is high, the method needs short time, energy consumption is low, and the method is a high-efficiency, green and convenient extraction method for the astragalus membranaceus polysaccharide and is suitable for large-scale industrial production. The method is expected to be used as an extraction method of novel traditional Chinese medicines and other natural medicines.

Description

Technical Field

[0001] The invention belongs to the technical field of Chinese medicinal material extraction, and specifically relates to a method for preparing an astragalus polysaccharide extract by using an enzyme / ultrasound-assisted natural low eutectic solvent. Background Art

[0002] Astragalus mongholicus is the root of Astragalus mongholicus or Astragalus membranaceus of the genus Astragalus in the Leguminosae family. It is a traditional Chinese herbal medicine and is described in the 2020 edition of the Chinese Pharmacopoeia. It is sweet in taste, slightly warm in nature, and enters the spleen and lung meridians. It has the effects of raising yang and lifting sunken, strengthening the spleen and replenishing the middle, strengthening the body's defense and strengthening the exterior, and detoxifying and regenerating muscles. Astragalus polysaccharide (APS) is one of the main active ingredients of Astragalus. Studies in recent years have shown that it has important biological activities such as antioxidant, antiviral and immunomodulatory. Traditional APS extraction methods are generally water extraction or water extraction after defatting, with low extraction rates and high energy requirements. It requires repeated extraction and repeated alcohol precipitation, which has the disadvantages of long extraction time and high energy consumption. There are also microbial fermentation extraction, microwave-assisted extraction, negative pressure cavitation extraction, pressurized hot water extraction, etc. Microbial fermentation extraction needs to solve the problem of excellent strains, and microwave and negative pressure cavitation methods have high process equipment requirements and are not suitable for large-scale production.

[0003] At present, reflux extraction and ethanol precipitation are still the most commonly used extraction methods in pharmacopoeia and industrial applications. However, these methods often have problems such as low APS extraction rate, long extraction time, easy deterioration of extracts, and complicated preparation process. Summary of the invention

[0004] The purpose of the present invention is to solve the above technical problems and provide a green and efficient enzyme-assisted natural deep eutectic solvent ultrasonic extraction method for astragalus polysaccharide, which can efficiently extract astragalus polysaccharide, among which choline chloride-lactic acid (ChCl-Lac) has the best extraction efficiency, and the extraction rate of astragalus polysaccharide reaches 143 mg / g defatted astragalus powder, which is 88.2% higher than that when water is used as solvent, and is significantly better than the traditional heated water extraction method. This method integrates natural deep eutectic solvent (NaDES) method, enzymatic extraction method, and ultrasonic-assisted extraction method as "enzyme-assisted-natural deep eutectic solvent-ultrasonic extraction method" to extract polysaccharide from Chinese medicinal material astragalus, with high extraction rate, short time required for the method, low energy consumption, and is a highly efficient, green and convenient extraction method for astragalus polysaccharide, and is expected to be used as a new extraction method for Chinese medicine and other natural medicines.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The invention provides a method for preparing an astragalus polysaccharide extract. The method comprises the following steps: adding defatted astragalus powder to a low eutectic solvent aqueous solution containing cellulase, ultrasonically extracting at 50-75°C for 15-60min, centrifuging, and collecting the supernatant for alcohol precipitation to obtain the astragalus polysaccharide; the low eutectic solvent in the low eutectic solvent aqueous solution comprises one of choline chloride and glycerol (ChCl-Gly), choline chloride and lactic acid (ChCl-Lac), and choline chloride and urea (ChCl-Ur).

[0007] Further, the low eutectic solvent includes: DES-1 (ChCl-Gly) prepared by mixing choline chloride (ChCl) and glycerol (Gly) in a molar ratio of 1:2 and heating at 80°C for 1h; DES-2 (ChCl-Lac) prepared by mixing choline chloride (ChCl) and lactic acid (Lac) in a molar ratio of 1:1 and heating at 80°C for 1h; DES-3 (ChCl-Ur) prepared by mixing choline chloride (ChCl) and urea (Ur) in a molar ratio of 1:2 and heating at 80°C for 1h.

[0008] Furthermore, the volume concentration of the deep eutectic solvent in the deep eutectic solvent aqueous solution is 5-25%, more preferably 10%.

[0009] Furthermore, the weight of the astragalus powder and the volume of the low eutectic solvent aqueous solution (solid-liquid ratio) is 1:15-50 g / mL, more preferably 1:30 g / mL.

[0010] Furthermore, the ratio of the amount of cellulase to the amount of astragalus powder used is 0.5-2 U / mg, more preferably 1 U / mg.

[0011] Furthermore, the ultrasonic power is 300W.

[0012] Furthermore, the extraction conditions are preferably: ultrasonic extraction for 30 min at 75° C. and 300 W. Under this condition, a higher extraction rate can be obtained with lower energy consumption.

[0013] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in: the present invention provides an efficient and green method for ultrasonic extraction of astragalus polysaccharides using enzyme-assisted natural low eutectic solvents, which can efficiently extract astragalus polysaccharides and is a new and efficient extraction method suitable for traditional Chinese medicine and other natural medicines. DETAILED DESCRIPTION

[0014] In order to better clarify and understand the purpose, process scheme and advantages of the present invention, the technical scheme and implementation method of the present invention are further described clearly, completely and in detail through specific examples. It should be noted that the embodiments described in the present invention are implemented on the premise of the technical scheme of the present invention, and detailed implementation methods and specific operation processes are given, but they are only part of the embodiments of the present invention, not all of the embodiments. The specific implementation methods described are limited to explaining and interpreting the present invention, and do not limit the present invention. Based on the embodiments in the present invention, all other implementation methods obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0015] The experimental methods and conditions used in the following examples are conventional methods and conventional conditions unless otherwise specified. The materials, reagents or instruments used in the examples, unless otherwise specified, can be obtained from commercial sources or prepared by conventional methods. The reaction conditions embodied in the content of the invention of the present invention can achieve the reaction and obtain the product with the expected effect. Due to space limitations, some embodiments are listed below to further illustrate the advantages of the technical solution of the present invention.

[0016] The astragalus used in the embodiments of the present invention was collected and prepared in accordance with the "Chinese Pharmacopoeia" (2020).

[0017] Example 1. DES-2 as solvent

[0018] Choline chloride and lactic acid were mixed in a molar ratio of 1:1, heated and stirred at 80°C for 1h to form a clear and uniform low eutectic solvent, which was recorded as DES-2. A certain amount of dried Astragalus was crushed and sieved (60 mesh), and 4 times the amount of 95% ethanol (w / v) was added. Reflux extraction was performed twice, each for 30 minutes, and the residue was collected by filtration, dried at 50°C, and collected and stored as defatted Astragalus powder. 50.0 mg of the above Astragalus powder was placed in a 2.0 mL centrifuge tube, 1 U / mg cellulase was added, and 1.5 mL of solvent (containing 10% DES-2 and 90% water) was added and mixed. In a 75°C water bath, ultrasonic extraction was performed with a power of 300W for 30 minutes, and inactivated in a 90°C water bath for 10 minutes. Centrifuge at 8000rpm / min for 10 minutes. Take 0.3 mL of the supernatant, add 5 times the amount of 95% ethanol, vortex for 15 seconds, refrigerate at 4°C for 12 hours, centrifuge at 8000 rpm / min for 10 minutes, discard the supernatant, and obtain the astragalus polysaccharide extract. The DNS (3,5-dinitrosalicylic acid) method measured the polysaccharide extraction rate in the overall extraction process to be 143 mg / g defatted astragalus powder.

[0019] The specific steps of the DNS method are as follows:

[0020] Add 6.3g DNS and 262.0mL of 2mol / L NaOH solution to 500mL of hot water solution containing 182.0g of sodium potassium tartrate, then add 5g of crystalline phenol and 5g of anhydrous sodium sulfite, stir to dissolve, cool and add distilled water to make up to 1000mL, which is the DNS color developing solution. Store in a brown bottle for later use.

[0021] The astragalus polysaccharide extract obtained above was dissolved in 1.5 mL of water to obtain a reducing sugar test solution; 0.3 mL of the reducing sugar test solution was accurately measured and placed in a centrifuge tube, 0.3 mL of 6 mol / L HCl solution was added, and a boiling water bath was placed for 30 min. The mixture was cooled to room temperature with running water, and 0.3 mL of 6 mol / L NaOH solution was added for neutralization. 0.6 mL of distilled water was added and mixed to obtain a total sugar test solution.

[0022] Accurately measure 0.5 mL of reducing sugar test solution and total sugar test solution, respectively, and place them in test tubes. Use 0.5 mL of water as a control. Add 0.5 mL of DNS reagent to each tube, mix well, boil in a boiling water bath for 5 min, cool to room temperature with running water, add 5.0 mL of water, mix well, and measure the absorbance values ​​at a wavelength of 540 nm. Calculate the mass of reducing sugar and total sugar in the sample using the standard curve prepared with glucose standard solution.

[0023] The extraction rate of astragalus polysaccharide extract = (total sugar mass - reducing sugar mass) / defatted astragalus mass, and the extraction rate (mg / g) was calculated.

[0024] Example 2. DES-1 as solvent

[0025] Choline chloride and glycerol were mixed in a molar ratio of 1:2, heated and stirred at 80℃ for 1h to form a clear and uniform low eutectic solvent, which was recorded as DES-1. 50.0mg defatted Astragalus powder was placed in a 2.0mL centrifuge tube, 1U / mg cellulase was added, and 1.5mL solvent (containing 10% DES-1 and 90% water) was added and mixed. In a 75℃ water bath, 300W power ultrasonic assisted extraction was performed for 30min, and inactivated in a 90℃ water bath for 10min. Centrifuged at 8000rpm / min for 10min. 0.3mL of supernatant was taken, 5 times the amount of 95% ethanol was added, vortexed for 15s, refrigerated at 4℃ for 12h, centrifuged at 8000rpm / min for 10min, and the supernatant was discarded to obtain Astragalus polysaccharide extract. The DNS method was used to determine that the polysaccharide extraction rate was 108mg / g defatted Astragalus.

[0026] Example 3. DES-3 as solvent

[0027] Choline chloride and urea were mixed in a molar ratio of 1:2, heated and stirred at 80℃ for 1h to form a clear and uniform low eutectic solvent, which was recorded as DES-3. 50.0mg defatted Astragalus powder was placed in a 2.0mL centrifuge tube, 1U / mg cellulase was added, and 1.5mL solvent (containing 10% DES-3 and 90% water) was added and mixed. In a 75℃ water bath, 300W power ultrasonic assisted extraction was performed for 30min, and inactivated in a 90℃ water bath for 10min. Centrifuged at 8000rpm / min for 10min. 0.3mL of supernatant was taken, 5 times the amount of 95% ethanol was added, vortexed for 15s, refrigerated at 4℃ for 12h, centrifuged at 8000rpm / min for 10min, and the supernatant was discarded to obtain Astragalus polysaccharide extract. The DNS method detected that the polysaccharide extraction rate was 103mg / g defatted Astragalus.

[0028] Example 4. DES-2 as solvent, scale-up experiment

[0029] Choline chloride and lactic acid were mixed in a molar ratio of 1:1, heated and stirred at 80°C for 1h to form a clear and uniform low eutectic solvent, which was recorded as DES-2. 1.0g defatted Astragalus powder was placed in a 50.0mL centrifuge tube, 1U / mg cellulase was added, and 30.0mL solvent (containing 10% DES-2 and 90% water) was added and mixed. In a 75°C water bath, 300W power ultrasonic assisted extraction was performed for 30min, and inactivated in a 90°C water bath for 10min. Centrifuged at 8000rpm / min for 10min. 0.3mL of supernatant was taken, 5 times the amount of 95% ethanol was added, vortexed for 15s, refrigerated at 4°C for precipitate for 12h, centrifuged at 8000rpm / min for 10min, and the supernatant was discarded to obtain an Astragalus polysaccharide extract. The DNS method detected that the polysaccharide extraction rate was 120mg / g defatted Astragalus. It can be seen that the method of the present invention can be well applied to the scale-up experiment and can still efficiently extract Astragalus polysaccharides.

[0030] Comparative Example 1 Conventional Heating Water Extraction

[0031] Take 50.0 mg defatted Astragalus powder and place it in a 2.0 mL centrifuge tube, add 1.5 mL of water, and extract in a 100 ° C water bath for 2 hours. Centrifuge at 8000 rpm / min for 10 minutes. Take 0.3 mL of the supernatant, add 5 times the amount of 95% ethanol, vortex for 15 seconds, refrigerate at 4 ° C for 12 hours, centrifuge at 8000 rpm / min for 10 minutes, discard the supernatant, and obtain the Astragalus polysaccharide extract. The polysaccharide extraction rate measured by DNS method is 76 mg / g defatted Astragalus.

[0032] Comparative Example 2 20% ethanol as solvent, ultrasonic extraction method

[0033] Take 50.0mg defatted Astragalus powder and place it in a 2.0mL centrifuge tube, add 1.5mL 20% ethanol solution, extract with 300W power ultrasound in a 75℃ water bath for 30min, and inactivate in a 90℃ water bath for 10min. Centrifuge at 8000rpm / min for 10min. Take 0.3mL of supernatant, add 5 times the amount of 95% ethanol, vortex for 15s, refrigerate at 4℃ for 12h, centrifuge at 8000rpm / min for 10min, discard the supernatant, and obtain Astragalus polysaccharide extract. The polysaccharide extraction rate measured by DNS method is 60mg / g defatted Astragalus.

[0034] Comparative Example 3DES-4 is a solvent

[0035] L-proline and lactic acid were mixed in a molar ratio of 1:1, heated and stirred at 80℃ for 1h to form a clear and uniform low eutectic solvent, which was recorded as DES-4. 500g of dried Astragalus was crushed and sieved (60 mesh), and 4 times the amount of 95% ethanol (w / v) was added. Reflux extraction was performed twice, each for 30min. After removing the fat-soluble components, the residue was dried at 50℃ and collected and stored. 50.0mg of Astragalus powder was placed in a 2.0mL centrifuge tube, 1U / mg cellulase was added, and 1.5mL of solvent (containing 10% DES-4 and 90% water) was added and mixed. In a 75℃ water bath, ultrasonic extraction was performed with a power of 300W for 30min, and inactivated in a 90℃ water bath for 10min. Centrifuge at 8000rpm / min for 10min. Take 0.3 mL of supernatant, add 5 times the amount of 95% ethanol, vortex for 15 seconds, refrigerate at 4°C for 12 hours, centrifuge at 8000 rpm / min for 10 minutes, discard the supernatant, and obtain astragalus polysaccharide. Add 1.5 mL of water to dissolve and obtain reducing sugar test solution. The DNS method was used to determine that the polysaccharide extraction rate was 68 mg / g defatted astragalus.

[0036] Comparative Example 4 DES-1 is the solvent (ultrasound-assisted extraction, no enzyme added)

[0037] Choline chloride and glycerol were mixed in a molar ratio of 1:2, heated and stirred at 80°C for 1h to form a clear and uniform low eutectic solvent, which was recorded as DES-1. 500g of dried Astragalus was crushed and sieved (60 mesh), and 4 times the amount of 95% ethanol (w / v) was added. Reflux extraction was performed twice, each for 30min. After removing the fat-soluble components, the residue was dried at 50°C and collected and stored. 50.0mg of Astragalus powder was placed in a 2.0mL centrifuge tube, 1.5mL of solvent (containing 10% DES-1 and 90% water) was added and mixed. In a 75°C water bath, ultrasonic assisted extraction was performed at 300W power for 30min, and inactivated in a 90°C water bath for 10min. Centrifuge at 8000rpm / min for 10min. Take 0.3 mL of supernatant, add 5 times the amount of 95% ethanol, vortex for 15 seconds, refrigerate at 4°C for 12 hours, centrifuge at 8000 rpm / min for 10 minutes, discard the supernatant, and obtain astragalus polysaccharide. Add 1.5 mL of water to dissolve and obtain reducing sugar test solution. The DNS method was used to determine that the polysaccharide extraction rate was 81 mg / g defatted astragalus.

[0038] Comparative Example 5 DES-1 is a solvent (enzymatic extraction without ultrasound assistance)

[0039] Choline chloride and glycerol were mixed in a molar ratio of 1:2, heated and stirred at 80℃ for 1h to form a clear and uniform low eutectic solvent, which was recorded as DES-1. 500g of dried Astragalus was crushed and sieved (60 mesh), and 4 times the amount of 95% ethanol (w / v) was added. Reflux extraction was performed twice, each for 30min. After removing the fat-soluble components, the residue was dried at 50℃ and collected for storage. 50.0mg of Astragalus powder was placed in a 2.0mL centrifuge tube, 1.5mL of solvent (containing 10% DES-1 and 90% water) was added and mixed. Extracted in a 75℃ water bath for 30min, and inactivated in a 90℃ water bath for 10min. Centrifuged at 8000rpm / min for 10min. Take 0.3mL of the supernatant, add 5 times the amount of 95% ethanol, vortex for 15s, refrigerate at 4℃ for 12h, centrifuge at 8000rpm / min for 10min, discard the supernatant, and obtain Astragalus polysaccharide. Add 1.5 mL of water to dissolve and obtain the reducing sugar test solution. The DNS method was used to determine that the polysaccharide extraction rate was 38 mg / g defatted astragalus.

[0040] From the above, it can be seen that the use of the low eutectic solvent described in the present invention, under the synergistic effect of ultrasound and enzyme, can achieve an excellent extraction rate of astragalus polysaccharides, and is significantly higher than the traditional heated water extraction method; and the method of the present invention requires a short time and low energy consumption, and is an efficient, green and convenient extraction method for astragalus polysaccharides.

[0041] The above-described embodiments are only preferred solutions of the present invention and are not intended to limit the present invention in any form. Other variations and modifications are possible without exceeding the technical solutions described in the claims.

Claims

1. A method for preparing an astragalus polysaccharide extract, characterized in that: The method comprises the following steps: adding defatted astragalus powder to a low eutectic solvent aqueous solution containing cellulase, ultrasonically extracting at 50-75°C for 15-60 minutes, centrifuging, and collecting the supernatant for alcohol precipitation to obtain astragalus polysaccharide; the low eutectic solvent in the low eutectic solvent aqueous solution comprises one of choline chloride and glycerol, choline chloride and lactic acid, and choline chloride and urea.

2. The method for preparing an astragalus polysaccharide extract according to claim 1, characterized in that: The low eutectic solvent includes: DES-1 prepared by mixing choline chloride and glycerol in a molar ratio of 1:2 and heating at 80°C for 1h; DES-2 prepared by mixing choline chloride and lactic acid in a molar ratio of 1:1 and heating at 80°C for 1h; DES-3 prepared by mixing choline chloride and urea in a molar ratio of 1:2 and heating at 80°C for 1h.

3. The method for preparing an astragalus polysaccharide extract according to claim 2, characterized in that: The low eutectic solvent is prepared by mixing choline chloride and lactic acid in a molar ratio of 1:1 and heating at 80° C. for 1 hour.

4. The method for preparing an astragalus polysaccharide extract according to claim 1, characterized in that: The volume concentration of the deep eutectic solvent in the deep eutectic solvent aqueous solution is 5-25%.

5. The method for preparing an astragalus polysaccharide extract according to claim 4, characterized in that: The volume concentration of the deep eutectic solvent in the deep eutectic solvent aqueous solution is 10%.

6. The method for preparing an astragalus polysaccharide extract according to claim 1, characterized in that: The weight of the astragalus powder and the volume of the low eutectic solvent aqueous solution are 1:15-50g / mL.

7. The method for preparing an astragalus polysaccharide extract according to claim 6, characterized in that: The weight of the astragalus powder and the volume of the low eutectic solvent aqueous solution are 1:30 g / mL.

8. The method for preparing an astragalus polysaccharide extract according to claim 1, characterized in that: The ratio of the cellulase amount to the astragalus powder is 0.5-2 U / mg.

9. The method for preparing an astragalus polysaccharide extract according to claim 8, characterized in that: The ratio of the cellulase amount to the astragalus powder is 1 U / mg.

10. The method for preparing an astragalus polysaccharide extract according to claim 1, characterized in that: The extraction conditions were ultrasonic extraction at 75°C and 300W for 30 min.