Method for extracting astragaloside IV

Abstract

The invention relates to a method for extracting astragaloside, which belongs to the field of natural medicines, and comprises the following steps of: crushing astragalus membranaceus, and performing reflux extraction in an alkaline solvent, the pH range of the alkaline environment is 9-13, and the pH range of the alkaline condition is regulated by using alkaline reagents KOH, NaOH and Na2CO3.

Description

Technical Field

[0001] This invention belongs to the field of natural medicines, and specifically relates to a method for extracting astragaloside A. Technical Background

[0002] Astragalus membranaceus (Fisch.), a plant belonging to the legume family, is a species of astragalus.

[0003] The dried roots of *Astragalus membranaceus* (Bge.) Hsiao or *Astragalus membranaceus* (Fisch.) Bge. are harvested in spring and autumn, with the fibrous roots and root heads removed before sun-drying. Astragalus is slightly warm in nature, sweet in taste, with a thin aroma but a strong flavor. It enters the lung and spleen meridians and has the functions of tonifying qi and strengthening the exterior, promoting diuresis and eliminating toxins, relieving pain and draining pus, and promoting wound healing and tissue regeneration. Its applications are very wide.

[0004] Astragalosides IV (ASI) is a tetracyclic triterpenoid compound with various pharmacological activities isolated from Astragalus membranaceus. The Chinese Pharmacopoeia and enterprise products use astragalosides IV as an indicator component for the quality testing of Astragalus membranaceus.

[0005] The 2020 edition of the Chinese Pharmacopoeia discloses the method for determining the content of astragaloside A: determination by high performance liquid chromatography (General Rule 0512).

[0006] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the stationary phase; acetonitrile-water (32:68) was used as the mobile phase; evaporative light scattering detector was used for detection. The theoretical plate number, calculated based on the astragaloside A peak, should not be less than 4000.

[0007] Preparation of reference solution: Take an appropriate amount of astragaloside A reference standard, accurately weigh it, and add 80% methanol to prepare a solution containing 0.5 mg per ml.

[0008] Preparation of the test solution: Accurately weigh about 1g of the powder (passed through a No. 4 sieve), place it in a stoppered conical flask, accurately add 50ml of 80% methanol solution containing 4% concentrated ammonia test solution (take 4ml of concentrated ammonia test solution, add 80% methanol to 100ml, and shake well), stopper tightly, weigh, heat under reflux for 1 hour, cool, weigh again, replenish the lost weight with 80% methanol solution containing 4% concentrated ammonia test solution, shake well, filter, accurately measure 25ml of the subsequent filtrate, evaporate to dryness, dissolve the residue in 80% methanol, transfer to a 5ml volumetric flask, add 80% methanol to the mark, shake well, filter, and collect the subsequent filtrate to obtain the test solution.

[0009] Determination method: Accurately pipette 2 μl (or 5 μl) and 10 μl of the reference solution and 10–20 μl of the test solution into the liquid chromatograph, respectively, and determine the result by calculating the logarithmic equation using the external standard two-point method.

[0010] However, since the content of astragaloside A in Astragalus membranaceus is low and it only has weak ultraviolet absorption at 200 nm, its content is usually determined by evaporative light scattering detector. However, the detection process is greatly affected by other components, and only a weak peak can be detected. This is not conducive to the quality monitoring of Astragalus membranaceus products. Therefore, the existing technology provides an improved analytical method for astragaloside A.

[0011] CN200610065282.9 discloses a method for quality detection of the fingerprint spectrum of astragaloside injection. The method uses octadecylsilane-bonded silica gel as the packing material; acetonitrile-water as the mobile phase for gradient elution; flow rate 0.5–1.0 ml / min; column temperature 20–40℃; an appropriate amount of astragaloside A reference standard is dissolved in methanol as a reference solution; the astragaloside injection is concentrated, extracted with water-saturated n-butanol as the extraction solvent, and impurities are removed with water saturated with n-butanol. After the water-saturated n-butanol extract is evaporated to dryness, methanol is added to the residue to dissolve it, and the methanol solution is used as the test solution for fingerprint spectrum detection of astragaloside injection. The fingerprint spectrum of the test solution is detected by high-performance liquid chromatography (HPLC). The fingerprint spectrum of the test sample should have good similarity to the reference fingerprint spectrum, with a similarity > 0.90.

[0012] CN202010862597.6 discloses a method and application for detecting components in Astragalus membranaceus and its preparations, including: detecting the test solution of Astragalus membranaceus or its preparations using a high-performance liquid chromatography-electrospray detector; calculating the content of flavonoids and / or saponins in the test solution using an external standard method or a one-test-multiple-evaluation method; the detection conditions include: gradient elution using mobile phase A and mobile phase B; mobile phase A is acetonitrile containing 0 vol.% to 0.1 vol.% formic acid, and mobile phase B is water containing 0 vol.% to 0.1 vol.% formic acid.

[0013] CN201711369841.X discloses a method for determining the content of astragaloside A in Kangai injection by HPLC-ELSD, including the following steps: (1) preparing a reference solution: taking astragaloside A reference standard and methanol to prepare a reference solution; (2) preparing a sample solution to be tested: accurately taking a sample of Kangai injection and diluting it with methanol to prepare a sample solution to be tested; (3) injecting the reference solution into a high-performance liquid chromatograph and an evaporative light scattering detector to establish a standard curve for astragaloside A; (4) injecting the sample solution to be tested into a high-performance liquid chromatograph. The peak area of ​​astragaloside A was determined by spectrometer and evaporative light scattering detector, and the content of astragaloside A was obtained according to the standard curve. The chromatographic conditions of steps (3) and (4) were as follows: 4.6 mm × 250 mm Shimadzu VP-ODS column; mobile phase: acetonitrile:water = 30:70; flow rate: 1.0 ml / min; column temperature: 30 ℃; injection volume: 10 μl; ELSD parameters: nebulizer temperature 60 ℃, drift tube temperature 89 ℃, nitrogen pressure 0.70 MPa, gas flow rate 1.15 L / min. The content of astragaloside A was calculated according to the regression equation. Three batches of Kangai injection with different batch numbers were tested, and the contents of astragaloside A were 0.09 mg / ml, 0.102 mg / ml, and 0.12 mg / ml, respectively.

[0014] Although existing technologies offer a variety of solutions, when conducting quality testing on Astragalus membranaceus, problems still exist, such as low astragaloside A content, significant interference from other components, small peaks in liquid chromatography, unobservable peak shapes, and low detection accuracy.

[0015] Therefore, this invention provides an HPLC method for determining the content of astragaloside A. Firstly, it utilizes an ODS column based on liquid-solid chromatography theory to improve the extraction efficiency of astragaloside A. This invention employs selective adsorption and selective elution of the sample to remove impurities from the target compound. Two common strategies are used: one is to adsorb interfering substances while the target component passes through the ODS column and is collected; the other is to adsorb the target component while interfering substances pass through the ODS column, with the target component ultimately eluting out. Since astragaloside A is present in relatively low amounts in Astragalus membranaceus, adsorption followed by elution is employed for astragaloside A. Summary of the Invention

[0016] This invention provides a method for extracting astragaloside A.

[0017] The alkaline conditions include a pH range of 9 to 13.

[0018] The alkaline conditions are adjusted using alkaline reagents KOH, NaOH, and Na2CO3.

[0019] The reflux extraction temperature is 70℃~100℃, the time is 1~5h, and the number of extractions is 1~3.

[0020] The reflux extraction solvent is a 70%–100% methanol solution, and the material-to-liquid ratio is 30–70 times the mass of Astragalus membranaceus.

[0021] This invention provides a method for removing astragaloside A from anaerobic digestion using ODS column adsorption. The method includes the following steps: dilution, sample loading, washing, elution, and concentration.

[0022] In the dilution step, deionized water is used to dilute the extract until the methanol concentration is 40% to 60%.

[0023] In the sample loading step, the diluted astragaloside A extract is loaded, and the loading amount is 1 / 5 to 5 times the ODS column packing volume.

[0024] In the washing and elution steps, the washing solution is a 2%–5% methanol solution, and the eluent is 70%–100% methanol. The volumes of both the washing solution and the eluent are 10–20 times the column volume.

[0025] In the concentration step, the concentration factor is 1 to 10 times.

[0026] The methanol in this invention can be replaced with ethanol.

[0027] The processing technology of Astragalus membranaceus in this invention is as follows: After harvesting fresh Astragalus membranaceus, remove the soil, spread wood ash on the drying ground, spread the Astragalus membranaceus flat in the wood ash, and dry it. When it is about 60-70% dry, sort it into grades and tie it into small bundles according to the grade. Then dry it until it is completely dry.

[0028] The processing technology of Astragalus membranaceus in this invention is as follows: After harvesting fresh Astragalus membranaceus, remove the soil, spread wood ash on the drying ground, spread the Astragalus membranaceus flat in the wood ash, and dry it. When it is about 60-70% dry, sort it into grades and tie it into small bundles according to grade. Spray wood ash on the small bundles and dry them thoroughly.

[0029] The processing technology of Astragalus membranaceus in this invention is as follows: Fresh or dried Astragalus membranaceus is sliced, stir-fried, and wood ash is added during the stir-frying process.

[0030] The processing method of Astragalus membranaceus in this invention is as follows: fresh or dried Astragalus membranaceus is soaked in a wood ash solution and then dried.

[0031] The Astragalus of this invention is used to prepare astragaloside A.

[0032] The method for determining the content of the active ingredient astragaloside A in the sample in this invention employs high performance liquid chromatography.

[0033] The conditions were as follows: octadecylsilane-bonded silica gel was used as the packing material; acetonitrile-water (38:62) was used as the mobile phase; isocratic elution was performed at a column temperature of 30℃ and a flow rate of 1 mL / min for 40 min; an evaporative light scattering detector was used; the gas flow rate was 2.5 L / min; the drift tube temperature was 105℃; the injection volume was 20 μm; and the theoretical column number, calculated based on the astragaloside A peak, should not be less than 4000.

[0034] Preparation of the control standard in the experiment:

[0035] Accurately weigh an appropriate amount of astragaloside A reference standard and add 80% methanol to prepare a solution containing 0.5 mg per mL.

[0036] Preparation of the test solution:

[0037] Weigh out Astragalus membranaceus slices, pulverize them, add 30-70 times the amount of 70%-100% methanol solution, adjust the pH to 9-13 with NaOH, KOH or Na2CO3, reflux at 70℃-100℃ for 3-5 hours, reflux 1-3 times, filter, store the filtrate, wash the filter residue with the extract, and combine the filtrate and washing liquid.

[0038] Astragaloside A removal: The obtained astragaloside A extract was diluted with deionized water to a methanol concentration of 40%–60% to obtain the sample loading solution; 1 / 5–5 times the volume of the extract of the ODS column packing was used for loading; after loading, the interfering substances of astragaloside A were washed with 10–20 times the volume of the ODS column and 2%–5% methanol solution as the washing buffer; then, astragaloside A was eluted with 10–20 times the volume of the ODS column and 70%–100% methanol as the eluent, and the astragaloside A eluent was collected; the obtained astragaloside A eluent was concentrated 1–10 times and filtered through a 0.22 μm microporous membrane to obtain a high performance liquid chromatography (HPLC) sample.

[0039] This invention can improve the peak area of ​​astragaloside A by improving the quality of the test sample solution and adjusting the concentration factor so that the peak area is within the linear range of the standard curve, while retaining astragaloside A in the sample. Impurities are removed by ODS column, improving peak shape and increasing determination accuracy.

[0040] To ensure that interfering substances do not affect the reflux extraction of astragaloside A, while simultaneously enhancing the subsequent impurity removal process of astragaloside A and optimizing the liquid chromatography spectrum, this invention selects alkaline reagents such as NaOH, KOH, or Na2CO3 to adjust the pH to 9–13 for reflux extraction. Under these conditions, the dissolution of astragaloside A can be accelerated, the extraction time shortened, and the extraction rate of astragaloside A increased.

[0041] The sample loading amount in this invention is determined based on the mass ratio of Astragalus membranaceus to ODS column packing material. A ratio of 1 / 5 to 5 times is preferred, as astragaloside A can be well adsorbed and eluted. If the ratio is too small, dead adsorption occurs, and astragaloside A is not easily eluted. If the ratio is too large, astragaloside A will no longer be adsorbed, and the excess will flow out of the column with the sample solution during the loading process without being adsorbed. Both of these factors will affect the accuracy of astragaloside A measurement.

[0042] In this invention, the obtained extract is diluted before loading. Astragaloside A is easily eluted at methanol concentrations greater than 70%, but not easily eluted at methanol concentrations of 40%–60%. Therefore, by diluting the extract to a methanol concentration of 40%–60%, astragaloside A can be adsorbed onto the ODS column. At the same time, components that are easily eluted at methanol concentrations of 40%–60% are not easily adsorbed by the ODS column and flow out directly with the loading solution during the loading process. This step combines the washing and loading steps into one, separating interfering substances during the loading process and simplifying the impurity removal steps.

[0043] This invention involves washing the sample with a 2%–5% methanol solution after loading to remove the alkaline reagent used in pH adjustment. This prevents the alkaline reagent from entering the chromatographic column during subsequent high-performance liquid chromatography (HPLC) determination, thus protecting the column and eluting away interference from highly polar components. Finally, elution with 70%–100% methanol removes impurities from astragaloside A, reducing interference from other interfering matrices in the HPLC chromatogram of astragaloside A.

[0044] The liquid chromatography spectrum of astragaloside A obtained by this invention is greatly improved, with less interference from other components. The spectrum is dominated by the astragaloside A peak, and the experiment has good stability, thus improving the accuracy of astragaloside A detection in Astragalus membranaceus.

[0045] In existing technology, astragalus is processed by removing residual stems and fibrous roots, cutting off the rhizome, spreading it flat in a drying yard to dry, and when it is about 60-70% dry, it is sorted and tied into small bundles according to grade, and then dried until completely dry.

[0046] This invention optimizes the sun-drying and drying method of Astragalus membranaceus using plant ash, achieving unexpected technical results and increasing the content of astragaloside A. Attached Figure Description

[0047] Figure 1 : Liquid phase diagram of the solution flowing out after loading the astragaloside A standard into an ODS column when the standard is dissolved in 80% methanol.

[0048] Peak 1 is the astragaloside A peak.

[0049] Figure 2 Comparison of phase diagrams of effluent and methanol wash liquids with different gradients.

[0050] From top to bottom, they are: effluent, 2% methanol solution, 10% methanol, 20% methanol, 40% methanol, 50% methanol, 60% methanol, 70% methanol, 80% methanol, 90% methanol, and 100% methanol.

[0051] Figure 3 Liquid phase diagram of fractional collection of eluent.

[0052] Figure 4 Liquid phase diagram of astragaloside A extracted from Astragalus membranaceus slices without passing through an ODS column.

[0053] Figure 5 Liquid phase diagram of astragaloside A extracted from Astragalus membranaceus slices after passing through an ODS column. Detailed Implementation

[0054] To make the present invention easier to understand, the present invention will be further described below with reference to specific embodiments. These embodiments are not intended to limit the present invention in any way. They are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Any modifications or changes to the present invention that are easily implemented by those skilled in the art without departing from the technical solution of the present invention will fall within the scope of the claims of the present invention.

[0055] Example 1: Investigation of alkaline reagents

[0056] Weigh out Astragalus membranaceus slices, pulverize them, add 50 times the amount of 70% methanol solution, and adjust the pH to 9-13 with NaOH, KOH, and Na2CO3. Reflux at 100℃ for 5 hours, repeat the reflux twice, filter, store the filtrate, wash the filter residue with the extract, and combine the filtrate and washing liquid.

[0057] Purification of astragaloside A: The obtained astragaloside A extract was diluted with deionized water to a methanol concentration of 40%; 5 times the volume of the extract was loaded onto the column; after loading, it was washed with 15 times the volume of 2% methanol solution as the washing solution; astragaloside A was eluted with 15 times the volume of 100% methanol as the eluent, and the astragaloside A eluent was collected; the obtained astragaloside A eluent was filtered through a 0.22 μm microporous membrane to obtain a high performance liquid chromatography (HPLC) sample.

[0058] The astragaloside A was removed using the method described above. The results are shown in Table 1.

[0059]

[0060] The results show that different alkaline reagents have little effect on the results. However, compared with no pH adjustment, the content determination results are higher under alkaline conditions after reflux extraction. This may be because alkaline reflux can accelerate the dissolution of astragaloside A, shorten the extraction time, and improve the extraction rate of astragaloside A.

[0061] The Chinese Pharmacopoeia 2020 edition stipulates that the content of astragaloside A shall not be less than 0.080%.

[0062] The astragaloside A content in the three batches of Kangai Injection under CN201711369841.X was 0.09 mg / ml, 0.102 mg / ml, and 0.12 mg / ml, respectively.

[0063] The application of the ODS column in this invention is different from that in CN201711369841.X, where the ODS column is a stationary phase.

[0064] This invention has achieved unexpected technical effects, and is low in cost and quick to implement.

[0065] Example 2: Investigation of different sample loading amounts

[0066] Weigh out Astragalus membranaceus slices, pulverize them, add 30 times the amount of 80% methanol solution, adjust the pH to 13 with KOH, reflux at 70℃ for 1 hour, repeat the reflux 3 times, filter, store the filtrate, wash the filter residue with the extract, and combine the filtrate and washing liquid.

[0067] Purification of Astragaloside A: The obtained astragaloside A extract was diluted with deionized water to a methanol concentration of 50% to obtain the sample loading solution; 1 / 10, 1 / 5, 2, 5, and 8 times the volume of ODS column packing were taken for loading; after loading, the sample was washed with 20 times the volume of ODS column 5% methanol solution as washing solution; astragaloside A was eluted with 20 times the volume of ODS column 70% methanol as eluent, and the astragaloside A eluent was collected; the obtained astragaloside A eluent was concentrated 10 times and filtered through a 0.22 μm microporous membrane to obtain a high performance liquid chromatography (HPLC) sample.

[0068] The astragaloside A was removed using the method described above. The results are shown in Table 2.

[0069]

[0070] The results show that if the sample amount is too small, there may be dead adsorption, making it difficult for astragaloside A to be eluted. However, if the sample amount is too large, it exceeds the maximum adsorption capacity, and some astragaloside A is not adsorbed and is eluted during the sample loading process, resulting in inaccurate content determination. Therefore, the astragalus extract with a volume of 1 / 5 to 5 times the column packing material was determined as the sample solution.

[0071] Example 3: Astragaloside A standard sample loading in this invention

[0072] 2 mg of astragaloside A standard was dissolved in 4 mL of 80% methanol and loaded onto an ODS column. Elution was performed sequentially with 10 column volumes of 2%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and pure methanol. The eluates were collected and filtered through a 0.22 μm microporous membrane. High-performance liquid chromatography (HPLC) was performed using an evaporative light scattering detector to obtain the HPLC gradients. Most of the astragaloside A was not adsorbed during loading, and a small amount of astragaloside A was found to elute in 70% methanol. (See attached image) Figure 1 and attached Figure 2

[0073] Subsequent experiments require diluting the astragaloside A extract to a methanol concentration of 40%–60%, and using a 70%–100% methanol solution as the eluent to elute the astragaloside A.

[0074] Example 4: Eluent Investigation

[0075] Weigh out Astragalus membranaceus slices, pulverize them, add 50 times the amount of 100% methanol solution, adjust the pH to 11 with NaOH, reflux at 70℃ for 4 hours, reflux twice, filter, store the filtrate, wash the filter residue with the extract, and combine the filtrate and washing liquid.

[0076] Purification of Astragaloside A: The obtained astragaloside A extract was diluted with deionized water to a methanol concentration of 50% to obtain the sample loading solution; 5 times the volume of the extract of ODS column packing was used for loading; after loading, it was washed with 20 times the volume of ODS column 2% methanol solution as washing solution; astragaloside A was eluted with 20 times the volume of ODS column 70% methanol as eluent, and the astragaloside A eluent was collected every 5 times, and filtered through a 0.22 μm microporous membrane to obtain the high performance liquid chromatography sample.

[0077] The astragaloside A content was measured to be 0.10%. No astragaloside A peak was observed at the 15th fraction, while a small peak was observed at the 10th fraction. Therefore, the optimal eluent concentration for astragaloside A was determined to be 10–20 times. (See attached image) Figure 3

[0078] Example 5

[0079] Comparison of astragaloside A liquid phase images before and after impurity removal from Astragalus membranaceus slices

[0080] Weigh out Astragalus membranaceus slices, pulverize them, add 70 times the amount of 80% methanol solution, adjust the pH to 9 with Na2CO3, reflux at 70℃ for 4 hours, reflux once, filter, store the filtrate, wash the filter residue with the extract, and combine the filtrate and washing liquid.

[0081] The obtained extract was divided into two portions. One portion was concentrated five times, and the other portion was used to remove impurities from astragaloside A. The astragaloside A extract was diluted with deionized water to a methanol concentration of 60% to obtain the sample loading solution. One-fifth of the volume of the extract was loaded onto the column. After loading, the sample was washed with 10 times the volume of the ODS column using 2% methanol solution as the washing buffer. Astragaloside A was eluted with 10 times the volume of the ODS column using 80% methanol as the eluent, and the astragaloside A eluent was collected. The obtained astragaloside A eluent was concentrated 10 times and filtered through a 0.22 μm microporous membrane to obtain a high-performance liquid chromatography (HPLC) sample.

[0082] The eluent and the unrefined concentrate were filtered together through a 0.22 μm microporous membrane and subjected to high-performance liquid chromatography (HPLC) under the above-mentioned HPLC conditions. The HPLC chromatogram is shown in the attached figure. Figure 4 and Figure 5 The comparison showed that after impurity removal, the astragaloside A peak was less affected by other components, and the liquid chromatography diagram was greatly improved.

[0083] The content determination results before and after impurity removal are shown in Table 2 below:

[0084] Before cleaning After removing impurities Astragaloside A content (%) 0.095 0.099

[0085] For samples that have not undergone ODS column purification, the astragaloside A peak is significantly affected by other matrices, impacting its accuracy. By using an ODS column to purify astragaloside A, the astragaloside A peak is greatly improved, and the astragaloside A measurement is slightly increased, thus optimizing the astragaloside A liquid chromatography pattern and enhancing data reliability.

[0086] Example 6:

[0087] After harvesting fresh astragalus, remove the soil and spread it out in a drying yard to dry. When it is about 60-70% dry, sort it into grades and tie it into small bundles according to grade. Then dry it completely.

[0088] Example 7:

[0089] After harvesting fresh astragalus, remove the soil, spread wood ash on the drying ground, spread the astragalus evenly on the wood ash, and dry it. When it is about 60-70% dry, sort it into grades and tie it into small bundles according to grade. Then dry it until it is completely dry.

[0090] Example 8:

[0091] After harvesting fresh astragalus, remove the soil, spread wood ash on the drying ground, spread the astragalus evenly on the wood ash, and dry it. When it is about 60-70% dry, sort it into grades and tie it into small bundles according to grade. Spray the small bundles with wood ash and dry them thoroughly.

[0092] Example 9:

[0093] In Example 6, dried Astragalus membranaceus was sliced ​​and stir-fried over a low flame with an appropriate amount of wood ash solution added during the stir-frying process.

[0094] The method for preparing a wood ash solution is as follows: dissolve 50 grams of wood ash in 1 kilogram of water.

[0095] Example 10:

[0096] In Example 7, dried Astragalus membranaceus was sliced ​​and stir-fried over a low flame with an appropriate amount of wood ash solution added during the stir-frying process.

[0097] The method for preparing a wood ash solution is as follows: dissolve 50 grams of wood ash in 1 kilogram of water.

[0098] Example 11:

[0099] Fresh astragalus root is soaked in a wood ash solution and then dried.

[0100] The method for preparing a wood ash solution is as follows: dissolve 50 grams of wood ash in 1 kilogram of water.

[0101] Example 12:

[0102] Dried Astragalus membranaceus is soaked in a wood ash solution and then dried.

[0103] The method for preparing a wood ash solution is as follows: dissolve 50 grams of wood ash in 1 kilogram of water.

[0104] Example 13:

[0105] Examples 6-12 of the present invention describe a method for determining the content of the active ingredient astragaloside A in samples using high performance liquid chromatography.

[0106] The conditions were as follows: octadecylsilane-bonded silica gel was used as the packing material; acetonitrile-water (38:62) was used as the mobile phase; isocratic elution was performed at a column temperature of 30℃ and a flow rate of 1 mL / min for 40 min; an evaporative light scattering detector was used; the gas flow rate was 2.5 L / min; the drift tube temperature was 105℃; the injection volume was 20 μm; and the theoretical column number, calculated based on the astragaloside A peak, should not be less than 4000.

[0107] Preparation of the control standard in the experiment:

[0108] Accurately weigh an appropriate amount of astragaloside A reference standard and add 80% methanol to prepare a solution containing 0.5 mg per mL.

[0109] Preparation of the test solution:

[0110] Weigh out Astragalus membranaceus slices, pulverize them, add 30-70 times the amount of 70%-100% methanol solution, adjust the pH to 9-13 with NaOH, KOH or Na2CO3, reflux at 70℃-100℃ for 3-5 hours, reflux 1-3 times, filter, store the filtrate, wash the filter residue with the extract, and combine the filtrate and washing liquid.

[0111] Astragaloside A removal: The obtained astragaloside A extract was diluted with deionized water to a methanol concentration of 40%–60% to obtain the sample loading solution; 1 / 5–5 times the volume of the extract of the ODS column packing was used for loading; after loading, the interfering substances of astragaloside A were washed with 10–20 times the volume of the ODS column and 2%–5% methanol solution as the washing buffer; then, astragaloside A was eluted with 10–20 times the volume of the ODS column and 70%–100% methanol as the eluent, and the astragaloside A eluent was collected; the obtained astragaloside A eluent was concentrated 1–10 times and filtered through a 0.22 μm microporous membrane to obtain a high performance liquid chromatography (HPLC) sample.

Claims

1. A method for extracting astragaloside A, characterized in that, After pulverizing Astragalus membranaceus, it was extracted by reflux under alkaline solvent. The alkaline environment included a pH range of 9-13, and the pH range of the alkaline conditions was adjusted by alkaline reagents KOH, NaOH, and Na2CO3.

2. A method for extracting astragaloside A, characterized in that, A method for removing impurities from astragaloside A using ODS column adsorption includes the following steps: dilution, sample loading, washing, elution, and concentration. The dilution step involves diluting the methanol concentration of the extract with deionized water to 40%–60%. The sample loading amount in the sample loading step is 1 / 5 to 5 times the amount of ODS column packing material; The eluent is 70%–100% methanol or 70%–100% ethanol; The volume of washing and elution solutions used is 10-20 times the column volume.

3. An Astragalus membranaceus according to claims 1-2, characterized in that, After harvesting fresh astragalus, remove the soil, spread wood ash on the drying ground, spread the astragalus evenly on the wood ash, and dry it. When it is about 60-70% dry, sort it into grades and tie it into small bundles according to grade. Then dry it until it is completely dry.

4. An Astragalus membranaceus according to claims 1-2, characterized in that, After harvesting fresh astragalus, remove the soil, spread wood ash on the drying ground, spread the astragalus evenly on the wood ash, and dry it. When it is about 60-70% dry, sort it into grades and tie it into small bundles according to grade. Spray the small bundles with wood ash and dry them thoroughly.

5. An Astragalus membranaceus according to claims 1-2, characterized in that, Fresh or dried astragalus root, sliced ​​and stir-fried, with wood ash added during the stir-frying process.

6. An Astragalus membranaceus according to claims 1-2, characterized in that, Fresh or dried Astragalus membranaceus is soaked in a wood ash solution and then dried.

7. Astragalus membranaceus according to claims 3-5 for preparing astragaloside A according to requirements 1-2.

Images