A method for extracting astragaloside

Abstract

The application discloses an astragaloside extraction method and belongs to the technical field of Chinese herbal medicine effective component extraction. The extraction method comprises the following steps: mixing astragalus with methanol and choline chloride, ultrasonic extraction, filtration, adding water into the filtrate to precipitate astragaloside, separating the precipitated solid, redissolving the solid with methanol, column chromatography, sequentially eluting with water, 20-30% methanol solution and methanol, collecting the methanol eluent, evaporating the solvent, and preparing astragaloside. The simple extraction method can extract high-purity astragaloside, the mixture of methanol and choline chloride is used as an extractant, the dissolution of astragaloside is greatly promoted, the dissolution of other interfering components is limited, high-purity astragaloside is obtained, and the difficulty of purifying astragaloside is reduced.

Description

Technical Field

[0001] This invention belongs to the field of extraction technology of effective components of traditional Chinese medicine, and specifically relates to a method for extracting astragaloside A. Background Technology

[0002] Astragalus is sweet and slightly warm in nature; it enters the spleen and lung meridians. It tonifies qi and nourishes blood, strengthens qi and consolidates the exterior. It can also restore pulse and prevent collapse, promote body fluid production and quench thirst, making it an excellent medicine for restoring vitality. It has good effects on physical weakness, lethargy, weakness in the limbs, or sudden shock. Raw astragalus can treat blood stasis, swelling of the hands and feet, and skin sores, mainly due to its qi-tonifying, exterior-consolidating, diuretic, and detoxifying effects. Honey-processed astragalus can alleviate symptoms such as insufficient vital energy, weakened organ function, spleen deficiency and fatigue, and anemia, thanks to its qi-tonifying, blood-nourishing, and spleen-strengthening effects. Modern pharmacological studies have found that astragalus can prevent and treat colds, treat viral myocarditis, prevent and treat childhood bronchial asthma, treat and alleviate chronic hepatitis B, and treat and alleviate chronic nephritis. It can also be used for vision recovery after retinal detachment surgery and for the prevention and treatment of cardiovascular and cerebrovascular diseases such as hypertension, arteriosclerosis, and hyperlipidemia.

[0003] Astragalus membranaceus (Huangqi) mainly comprises saponins, polysaccharides, flavonoids, and organic acids. Saponins and astragalus polysaccharides are its main active substances, with astragaloside I, II, III, IV, V, VI, VII, and VIII being the primary saponin compounds. The 2020 edition of the Chinese Pharmacopoeia stipulates that the content of astragaloside IV is a key indicator for the quality control of astragalus. Astragaloside IV is a triterpenoid saponin found in genuine astragalus, and its content in astragalus is extremely low, generally around one-thousandth. Furthermore, the complex composition of astragalus makes extraction difficult. The development of modern pharmacology and the continuous emergence of new dosage forms and products necessitate the effective extraction of astragaloside IV from astragalus, which plays a crucial role in product research and innovation. Current research on extraction methods and processes for astragaloside IV has made some progress. Based on traditional extraction methods, supercritical carbon dioxide extraction, microwave-assisted extraction, and ultrasonic-assisted extraction technologies have emerged. Therefore, researching efficient and rapid methods and technologies for extracting astragaloside IV from astragalus is particularly important.

[0004] Modern medical research has proven that astragaloside A has effects such as reducing thrombus formation, lowering blood pressure, protecting the heart, and enhancing the body's immunity. Astragalus contains numerous chemical components, mainly saponins, flavonoids, and polysaccharides. Among these, saponins include astragaloside and daidzein, while flavonoids include four main categories: flavonoids, isoflavones, isoflavones, and pterostilbene. It also contains monosaccharides, amino acids, proteins, riboflavin, folic acid, nicotinic acid, vitamin D, oleic acid, linolenic acid, trace elements, vanillic acid, ferulic acid, isoflavonic acid, p-hydroxyphenylacrylic acid, caffeic acid, chlorogenic acid, palmitic acid, β-sitosterol, carotene, lupeol, and n-hexadecyl alcohol. Studies have shown that saponins are an important component of the pharmacodynamic material basis of astragalus, while astragaloside A is one of the main active ingredients of astragalus. How to efficiently extract astragaloside A from astragalus is a new direction for further research in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a method for extracting astragaloside A. Astragaloside A is a monomeric compound extracted and isolated from Astragalus membranaceus. It is a highly polar saponin, slightly soluble in methanol, almost insoluble in ethyl acetate, acetone, and water, and dissolves in ethanol upon heating, precipitating upon cooling. This invention involves ultrasonic extraction of Astragalus membranaceus with methanol and choline chloride. Choline chloride enhances the solubility of astragaloside A in methanol, making it easier to dissolve and increasing its yield. Water is then added to the extract to precipitate astragaloside A, which is further separated by column chromatography to obtain high-purity astragaloside A.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for extracting astragaloside A includes the following steps:

[0008] Astragalus membranaceus was mixed with methanol and choline chloride, extracted by ultrasonication, filtered, and water was added to the filtrate to precipitate astragaloside A. The precipitated solid was separated, redissolved with methanol, loaded onto a column, and eluted successively with water, a 20-30% (v / v) methanol solution, and methanol. The methanol eluent was collected, and the solvent was evaporated to dryness to obtain astragaloside A.

[0009] This invention uses methanol and choline chloride to prepare the extraction solution. Choline chloride acts as a hydrogen bond acceptor, which helps to separate astragaloside A from astragalus. Methanol is also a good solvent for astragaloside A. The combination of the two can greatly enhance the dissolution of astragaloside A. At the same time, since the extraction solution does not contain water, water-soluble components, such as polysaccharides, will not be extracted, which can reduce the difficulty of subsequent purification and obtain astragaloside A with higher purity.

[0010] Preferably, the molar ratio of methanol to choline chloride is (1-2):(0.1-0.2).

[0011] Preferably, the astragalus is dried astragalus powder.

[0012] The extracted astragalus can be either fresh or dried powder, with dried powder being preferred.

[0013] More preferably, the mass-to-volume ratio of Astragalus membranaceus to methanol is 1 g:(5-10) mL.

[0014] The extraction liquid volume selected in this invention can ensure the full dissolution of astragaloside A while avoiding waste caused by excessive solvent volume.

[0015] Preferably, the ultrasonic extraction uses an ultrasonic frequency of 20–25 kHz and a duration of 30–40 min.

[0016] When the ultrasonic frequency is between 20 and 25 kHz, it can maximize the breakage of Astragalus membranaceus tissue. Too high or too low a frequency is not conducive to the destruction of Astragalus membranaceus tissue. An ultrasonic time of 30 to 40 minutes can ensure the full dissolution of astragaloside A. If the time is too short, the dissolution will be incomplete, and if the time is too long, the amount of astragaloside A dissolved will not increase.

[0017] Preferably, the specific operation of adding water to the filtrate is to add 6 to 8 times the volume of water to the filtrate after ultrasonic extraction.

[0018] This invention reduces the concentration of methanol by adding water, turning it into a dilute methanol-water solution. This allows components that are readily soluble in methanol but insoluble or slightly soluble in water to be extracted. After reconstitution, the components are then subjected to gradient elution on a chromatographic column to obtain high-purity astragaloside A.

[0019] Preferably, the upper column is an upper CAD-40 macroporous resin column.

[0020] The beneficial technical effects of the present invention are as follows:

[0021] This invention provides a simple extraction method to obtain high-purity astragaloside A. Using a mixture of methanol and choline chloride as the extraction solvent, the extraction of astragaloside A is greatly promoted, while the extraction of other interfering components is limited. This reduces the difficulty of purifying astragaloside A while obtaining high-purity astragaloside A.

[0022] The preparation method provided by this invention can provide a material basis for expanding the application of astragaloside A, which is of great significance. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0024] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] Example 1

[0028] Extraction of astragaloside A:

[0029] The dried Astragalus membranaceus was pulverized using a plant pulverizer and passed through a 100-mesh sieve. 10g of the sieved Astragaloside A powder was weighed into a 100mL beaker, and 50mL of methanol (1.23mol) and 16.8g of choline chloride (0.12mol) were added. The mixture was stirred for 1min to ensure homogeneity. The mixture was then placed in an ultrasonic cleaner and ultrasonically extracted at a frequency of 20kHz for 30min. After filtration, 8 times the volume of water was added to the filtrate, and the mixture was allowed to stand for 24h to precipitate Astragaloside A. After filtration, the separated solid was redissolved in 5mL of methanol and loaded onto a column (pre-wetted with water using a CAD-40 macroporous resin chromatography column). The column was eluted first with 50mL of water, then with 40mL of 20% methanol solution, and finally with 40mL of methanol. The methanol eluent was collected, and the solvent was evaporated to dryness to obtain Astragaloside A.

[0030] Example 2

[0031] Extraction of astragaloside A:

[0032] The dried Astragalus membranaceus was pulverized using a plant pulverizer and passed through a 100-mesh sieve. 10g of the sieved Astragaloside A powder was weighed into a 100mL beaker, and 70mL of methanol (1.73mol) and 23.7g of choline chloride (0.17mol) were added. The mixture was stirred for 1min to ensure homogeneity. The mixture was then placed in an ultrasonic cleaner and ultrasonically extracted at a frequency of 25kHz for 40min. After filtration, 6 times the volume of water was added to the filtrate, and the mixture was allowed to stand for 24h to precipitate Astragaloside A. After filtration, the separated solid was redissolved in 5mL of methanol and loaded onto a column (pre-wetted with water using a CAD-40 macroporous resin chromatography column). The column was eluted first with 50mL of water, then with 40mL of 30% methanol solution, and finally with 40mL of methanol. The methanol eluent was collected, and the solvent was evaporated to dryness to obtain Astragaloside A.

[0033] Comparative Example 1

[0034] Extraction of astragaloside A:

[0035] The dried Astragalus membranaceus was pulverized using a plant pulverizer and passed through a 100-mesh sieve. 10g of the sieved Astragaloside A powder was weighed into a 100mL beaker, and 50mL of methanol was added. The mixture was stirred for 1 minute to ensure homogeneity. The mixture was then placed in an ultrasonic cleaner and ultrasonically extracted at a frequency of 20kHz for 30 minutes. After filtration, 8 times the volume of water was added to the filtrate, and the mixture was allowed to stand for 24 hours to allow Astragaloside A to precipitate. After filtration, the separated solid was redissolved in 5mL of methanol and loaded onto a column (a CAD-40 macroporous resin chromatography column pre-wetted with water). The column was eluted first with 50mL of water, then with 40mL of 20% methanol solution, and finally with 40mL of methanol. The methanol eluent was collected, and the solvent was evaporated to dryness to obtain Astragaloside A.

[0036] Comparative Example 2

[0037] Extraction of astragaloside A:

[0038] The dried Astragalus membranaceus was pulverized using a plant pulverizer and passed through a 100-mesh sieve. 10g of the sieved Astragaloside A powder was weighed into a 100mL beaker, and 50mL of methanol (1.23mol) and 16.8g of choline chloride (0.12mol) were added. The mixture was stirred for 1min to ensure homogeneity. The mixture was then placed in an ultrasonic cleaner and ultrasonically extracted at a frequency of 100kHz for 120min. After filtration, 8 times the volume of water was added to the filtrate, and the mixture was allowed to stand for 24h to precipitate Astragaloside A. After filtration, the separated solid was redissolved in 5mL of methanol and loaded onto a column (pre-wetted with water using a CAD-40 macroporous resin chromatography column). The column was eluted first with 50mL of water, then with 40mL of 20% methanol solution, and finally with 40mL of methanol. The methanol eluent was collected, and the solvent was evaporated to dryness to obtain Astragaloside A.

[0039] Comparative Example 3

[0040] Extraction of astragaloside A:

[0041] The dried Astragalus membranaceus was pulverized using a plant pulverizer and passed through a 100-mesh sieve. 10g of the sieved Astragaloside A powder was weighed into a 100mL beaker, and 50mL of methanol (1.23mol) and 16.8g of choline chloride (0.12mol) were added. The mixture was stirred for 1min to ensure homogeneity. The mixture was then placed in an ultrasonic cleaner and ultrasonically extracted at a frequency of 20kHz for 30min. After filtration, 8 times the volume of water was added to the filtrate, and the mixture was allowed to stand for 24h to allow Astragaloside A to precipitate. After filtration, the separated solid was washed with water and dried to obtain Astragaloside A.

[0042] For the astragaloside A extracted in Examples 1-2 and Comparative Examples 1-3, the samples were first weighed, and then their purity was determined by high-performance liquid chromatography (HPLC). The extraction yield of astragaloside A was calculated, and the results are shown in Table 1. Astragaloside A purchased from Chengdu Mansite Biotechnology Co., Ltd. was used as a reference standard for HPLC.

[0043] Extraction amount of astragaloside A (mg·g) -1 = Final extract weight (mg) × Purity (%) / Astragalus weight (g)

[0044] Table 1

[0045]

[0046] As can be seen from Table 1, the extraction rate of astragaloside A was significantly improved after the addition of choline chloride; the extension of ultrasonic frequency and ultrasonic time did not improve the extraction rate of astragaloside A; the column chromatography step selected in this invention can further improve the purity of astragaloside A, and the loss of astragaloside A is small.

[0047] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for extracting astragaloside A, characterized in that, Includes the following steps: Astragalus membranaceus was mixed with methanol and choline chloride, extracted by ultrasonication, filtered, and water was added to the filtrate to precipitate astragaloside A. The precipitated solid was separated, redissolved with methanol, loaded onto a column, and eluted successively with water, a 20-30% (v / v) methanol solution, and methanol. The methanol eluent was collected, and the solvent was evaporated to dryness to obtain astragaloside A. The molar ratio of methanol to choline chloride is (1~2):(0.1~0.2); The mass-to-volume ratio of Astragalus membranaceus to methanol is 1g:(5~10)mL; The ultrasonic extraction was performed at a frequency of 20-25 kHz for 30-40 minutes.

2. The extraction method of astragaloside A according to claim 1, characterized in that, The astragalus mentioned is dried astragalus powder.

3. The extraction method of astragaloside A according to claim 1, characterized in that, The specific procedure for adding water to the filtrate is to add 6 to 8 times the volume of water to the filtrate after ultrasonic extraction.

4. The extraction method of astragaloside A according to claim 1, characterized in that, The upper column is a CAD-40 macroporous resin column.

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