Method for extracting astragaloside IV

Abstract

The invention belongs to the technical field of traditional Chinese medicine processing, and particularly relates to an astragaloside extraction method. The active ingredient astragaloside IV of astragalus membranaceus is directly extracted by using a natural deep-eutectic solvent extraction method, betaine and lactic acid are mixed and dissolved according to the mass ratio of 5: (25-35) to obtain a lactic acid-betaine deep-eutectic solvent, astragalus membranaceus powder is added, extraction is performed through specific process parameters, the content of astragaloside IV in the extracted product is up to 4.2 mg / g, and the content of astragaloside IV in the extracted product is up to 4.2 mg / g. Compared with a traditional ethanol extraction method, the extraction rate is increased by 25%. The method has the advantages of simple and rapid process flow and easiness in industrial production.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine processing, and more particularly to a method for extracting astragaloside IV. BACKGROUND

[0002] Radix Astragali is a perennial medicinal herb of Leguminosae, which has the clinical effects of tonifying qi and ascending yang, stopping sweating and consolidating the exterior, promoting diuresis and removing edema, and is used for the treatment of deficiency of qi and fatigue, middle-qi deficiency, spontaneous sweating due to deficiency of qi, edema due to deficiency of qi, and emaciation due to deficiency of blood. Radix Astragali has been widely used in clinical medicine as an immune promoter or immune regulator, and can also be used as a feed additive to improve the growth performance of animals, enhance the immune function of various animals, and improve meat quality and flavor, delay the degree of rancidity. Radix Astragali contains many chemical components, including polysaccharides, saponins, flavonoids, and amino acids. Astragaloside IV is one of the main active components in Radix Astragali, and is an important index component for evaluating the quality of Radix Astragali and Radix Astragali preparations. Astragaloside IV has the effects of enhancing immune function, anti-inflammation, lowering blood pressure, stabilizing red blood cell membranes, increasing the content of plasma cyclic adenosine monophosphate (cAMP), and promoting DNA synthesis in regenerating livers of mice.

[0003] Traditional extraction and separation methods include decoction, cold immersion, reflux, and Soxhlet extraction. Currently, the extraction methods for astragaloside IV include water extraction, alkaline water extraction, supercritical fluid extraction, and ultrasonic-assisted extraction.

[0004] Water extraction of astragaloside IV is a relatively traditional extraction process, which can be divided into water extraction and alcohol extraction according to the extraction medium, and decoction and reflux according to the extraction method. The factors affecting the extraction rate of astragaloside IV by water extraction include extraction temperature, time, solid-liquid ratio, and number of times. However, water extraction of astragaloside IV is time-consuming and has a low extraction rate.

[0005] Alkaline water extraction is a method in which alkali is added during the extraction of astragaloside IV to convert some ring astragalol saponins into astragaloside IV, thereby improving the extraction rate of astragaloside IV. However, this method is complicated and wastes a lot of medicinal materials, and flavonoids and polysaccharides have certain effects on the extraction of astragaloside IV.

[0006] Ultrasonic-assisted extraction is a method in which ethanol is soaked after water extraction, followed by ultrasonic extraction and extraction with n-butanol. This method can shorten the extraction time and improve the extraction rate, but n-butanol has a high boiling point and is difficult to recover.

[0007] Supercritical fluid extraction technology is a new extraction and separation technology, with extraction temperature, extraction pressure, extraction time, extraction agent flow rate as observation factors. It has the characteristics of low operation temperature, good selectivity, one-step completion from extraction to separation, no solvent residue in product, etc. and has been applied in the fields of biological medicine, food, etc. Although the extraction rate of supercritical fluid extraction is higher than that of general extraction method, because the cost of its instrument equipment itself is high, the use and maintenance cost is also high, and the industrialized production still needs to further improve the process method.

[0008] In summary, the traditional astragaloside extraction method relies on organic solvents such as ethanol and methanol, and has problems such as toxicity residue, low extraction efficiency, high energy consumption, and destruction of heat-sensitive components. The current technology, including ultrasonic-assisted extraction and supercritical fluid extraction, still has the problem of high solvent and equipment cost. SUMMARY

[0009] In view of the above technical problems, the present application provides a method for extracting astragaloside.

[0010] The present application specifically adopts the following technical solutions: The present application provides a method for extracting astragaloside, comprising the following steps: S1, mixing and dissolving betaine and lactic acid to obtain a lactic acid-betaine eutectic solvent in a molar ratio of 0.5-1.5:2-4, mixing the lactic acid-betaine eutectic solvent with radix astragali powder in a mass-volume ratio of 0.5-1.5g:5-15mL, and then water bath oscillation extraction to obtain an extract; S2, centrifuging the extract to collect the supernatant containing astragaloside, and then vacuum distillation of the supernatant to recover the lactic acid-betaine eutectic solvent, and the obtained residue is the astragaloside extract.

[0011] The present application provides a method for extracting astragaloside, which is treated by a lactic acid-betaine eutectic solvent, and the extraction efficiency and product quality are significantly improved. Astragaloside mainly exists in the cell wall, and the lactic acid-betaine eutectic solvent mainly destroys the plant cell wall through hydrogen bond network, promotes the dissolution of astragaloside, and increases the extraction efficiency. Moreover, the extraction process has low energy consumption, the solvent can be recovered, the process is simple, and harmful solvents such as acid, base and methanol do not need to be added to the system, which has the advantages of simplicity, rapidness, greenness and easy industrialization.

[0012] Further, the dissolving conditions in S1 are 70-90℃ water bath magnetic stirring for 25-35 minutes.

[0013] Further, the temperature of the water bath oscillation is 55-65℃.

[0014] Further, the oscillation speed is 110-130rpm / min.

[0015] Further, the extraction time is 1-3h.

[0016] Further, the condition for removing the excessive lactic acid-beta alanine eutectic solvent is 40-60 DEG C, 0.05-0.15 MPa reduced pressure distillation.

[0017] Further, the centrifugal condition is 3000-5000 rpm centrifugation for 10-20 minutes.

[0018] Further, the viscosity of the lactic acid-beta alanine eutectic solvent is 400-600 mPa s.

[0019] The present application has the following beneficial effects: The present application uses a specific proportion of lactic acid-beta alanine eutectic solvent to improve the extraction efficiency of astragaloside, so that the target yield is greater than or equal to 90%, the energy consumption of the extraction process is reduced, and the solvent is efficiently recovered. The process flow of the method is simple, and harmful solvents such as acid, alkali and methanol do not need to be added to the system, and the method has the advantages of simplicity, rapidness, greenness and easy industrialization. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 It is a DPPH free radical clearance statistical graph under different molar ratios.

[0021] Figure 2 It is a DPPH free radical clearance statistical graph under different material liquid ratios.

[0022] Figure 3 It is a DPPH free radical clearance statistical graph under different extraction temperatures.

[0023] Figure 4 It is a DPPH free radical clearance statistical graph under different extraction times. DETAILED DESCRIPTION

[0024] The present application will be described in detail below in combination with the drawings and specific embodiments, but should not be understood as limiting the present application. If not specially stated, the technical means used in the following examples are conventional means familiar to those skilled in the art, and the materials, reagents, etc. used in the following examples, if not specially stated, can be obtained from commercial channels.

[0025] Example 1: Preparation of astragaloside extract.

[0026] I. Extraction method of astragaloside.

[0027] 1. Preparation of lactic acid-beta alanine eutectic solvent Weigh betaine and lactic acid at a molar ratio of 1:3. Add the betaine and lactic acid to a round-bottom flask and stir magnetically in an 80°C water bath for 30 minutes until a homogeneous and transparent lactic acid-betaine eutectic solvent with a viscosity of about 500 mPa·s is formed. Cool to room temperature for later use.

[0028] 2. Extraction of active ingredients from Astragalus membranaceus Weigh 5g of Astragalus powder that has passed through a 60-mesh sieve and mix it with lactic acid-betaine eutectic solvent at a mass-volume ratio of 1g:10mL, i.e., add 50mL of lactic acid-betaine eutectic solvent to obtain the extract. Extract the extract by shaking in a 60℃ constant temperature water bath for 2 hours (shaking frequency 120rpm / min). The hydrogen bond network of the lactic acid-betaine eutectic solvent is used to disrupt the plant cell wall and promote the dissolution of astragaloside A.

[0029] 3. Solid-liquid separation and solvent recovery The extract was centrifuged at 4000 rpm for 15 minutes, and the supernatant containing astragaloside A was collected. The supernatant was then distilled under reduced pressure at 50℃ and 0.1 MPa to recover the lactic acid-betaine eutectic solvent. The recovered residue was dissolved in 100 mL of deionized water and filtered through a 0.45 μm microporous membrane to obtain the astragaloside A extract.

[0030] II. Ethanol extraction method.

[0031] 1. Weigh 30g of Astragalus powder and 70% ethanol at a mass-volume ratio of 1g:10mL, mix and soak for 10h, then extract by reflux in a constant temperature water bath at 90°C for 1 hour using a reflux condenser. Transfer the extracted extract to an Erlenmeyer flask for later use.

[0032] III. Efficacy determination of Astragalus extract.

[0033] 1. Determination of DPPH free radical scavenging rate The antioxidant activity of the purified Astragalus membranaceus extract was determined. The DPPH free radical scavenging rate of the extract was detected by spectrophotometry. The absorbance was measured at a wavelength of 517 nm, and the scavenging rate was calculated using the following formula: Clearance rate (%) = [A0 - (A0)] x -A x0 )] / A0×100% A0 represents the absorbance value of the model control group (where the sample solution was replaced with an equal volume of anhydrous ethanol). x Indicates the absorbance value of the sample solution; A X0 This indicates the absorbance value of the sample control group (where the anhydrous ethanol solution of DPPH was replaced with an equal amount of anhydrous ethanol).

[0034] The experimental results are shown in Table 1. The DPPH scavenging rate indicates the extract's ability to capture free radicals. The scavenging rate of Astragalus extract extracted by lactic acid-betaine eutectic solvent reached 36.23%, which was significantly higher than that of ethanol extract. This is attributed to the high efficiency of the lactic acid-betaine eutectic solvent system in dissolving antioxidant components such as polyphenols and flavonoids in Astragalus.

[0035] 2. Total antioxidant capacity determination The total antioxidant capacity (T-AOC) of Astragalus membranaceus extract was determined using the ferric reducing power (FRAP) method. The absorbance was measured at 596 nm, and the total antioxidant capacity was calculated using the following formula: The standard curve for ferrous sulfate is y = 0.674x + 0.1492, R0 2 =0.9979, (y is net absorbance, x is Fe²⁺ concentration) Equivalent Fe²⁺ concentration in the reaction system: Csystem = (ysample - 0.1492) / 0.674 (ysample is the absorbance of the sample) Equivalent Fe²⁺ concentration of the original sample solution: Coriginal = Csystem × Vtotal / Vsample ÷ D (Vtotal is the total volume of the reaction system, μL; Vsample is the sample loading volume, μL; D is the sample dilution factor;) Total antioxidant capacity of liquid sample (μmol Fe²⁺ equivalent / mL) = Coriginal × 10⁻³ Total antioxidant capacity of solid sample (μmol Fe²⁺ equivalent / g) = Coriginal × 10⁻³ × Vextracted / msample (Vextracted is the total extract volume, mL; msample is the sample mass, g) The experimental results are shown in Table 1. The total antioxidant capacity reflects the electron transfer capacity of the extract. The antioxidant activity of the extract of this invention is enhanced, and the total antioxidant capacity reaches 36.69 mmol Fe. 2+ The equivalent / mL test verified that the lactic acid-betaine eutectic solvent extraction process has a better retention effect on active ingredients, while traditional high-temperature ethanol extraction is prone to degradation of heat-sensitive antioxidant components.

[0036] Table 1: Comparison of the efficacy of extracts 3. Determination of Astragaloside A Content Accurately weigh 10.0 mg of astragaloside A reference standard and dilute to 10 mL with methanol to obtain a 1.0 mg / mL stock solution. Take 0.05, 0.1, 0.2, 0.5, and 1.0 mL of the stock solution and dilute to 10 mL with the mobile phase to obtain a series of solutions with concentrations of 0.005–0.1 mg / mL. Filter the solutions through a 0.22 μm organic filter membrane.

[0037] System suitability test: Inject 0.02 mg / mL reference solution 5 times consecutively, record peak area, RSD≤2.0%, astragaloside theoretical plate number≥3000, resolution≥1.5.

[0038] Chromatographic detection: Chromatographic conditions were set as follows: C18 column (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile-water (30:70); flow rate: 1.0 mL / min; column temperature: 30 °C; injection volume: 10 μL; evaporative light scattering detector (drift tube 80–100 °C, carrier gas 2.0 L / min). The reference solution series and sample solution were injected separately, and the peak areas were recorded.

[0039] The astragaloside A content was found to be 4.2 mg / g by HPLC, which is 25% higher than that of the traditional ethanol extraction method.

[0040] Verification Example 1: Optimization of extraction conditions.

[0041] 1. Optimization of the molar ratio of lactic acid and betaine To demonstrate the effect of the molar ratio of lactic acid to betaine on the DPPH radical scavenging rate, the following experimental treatments were conducted, with influencing factors including molar ratios of betaine to lactic acid of 1:1, 1:2, 1:3, 1:4, and 1:5. The remaining steps were the same as in Example 1.

[0042] 2. Optimization of feed-liquid ratio conditions To demonstrate the effect of the feed-to-liquid ratio on the DPPH free radical scavenging rate, the following experimental treatments were conducted, with the influencing factors including: the mass-to-volume ratio of astragalus powder to lactic acid-betaine eutectic solvent of 1g:5mL, 1g:10mL, 1g:15mL, 1g:20mL, and 1g:25mL, respectively. The remaining steps were the same as in Example 1.

[0043] 3. Optimization of extraction temperature conditions To demonstrate the effect of extraction temperature on DPPH radical scavenging rate, the following experimental treatment was conducted, with influencing factors including constant temperature water bath temperatures of 40℃, 50℃, 60℃, 70℃, and 80℃. The remaining steps were the same as in Example 1.

[0044] 4. Optimized extraction time To demonstrate the effect of extraction time on DPPH radical scavenging rate, the following experimental treatments were conducted, with influencing factors including constant temperature water bath extraction times of 60 min, 90 min, 120 min, 150 min, and 180 min. The remaining steps were the same as in Example 1.

[0045] 5. Experimental Results Single-factor influence curves were plotted by measuring the DPPH free radical scavenging rate at different levels. Experimental results are as follows: Figures 1-4As shown, in the molar ratio experiment, the removal rate was optimal at 1:3, and the removal rate increased or decreased after deviating from this ratio; in the material-liquid ratio experiment, the extraction effect was best at 1:10; the removal rate peaked when the extraction temperature reached 60℃; the efficiency inflection point was 120 min after extraction, and the effect of extending the extraction time slowed down.

[0046] In summary, the optimal single-factor parameters for lactic acid-betaine eutectic solvent extraction are: molar ratio 1:3, material-liquid ratio 1:10, extraction temperature 60℃, and extraction time 120 min. Under these conditions, the DPPH free radical scavenging rate is the highest, and the extraction effect is the best.

[0047] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0048] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0049] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for extracting astragaloside A, characterized in that, Includes the following steps: S1. Betaine and lactic acid are mixed and dissolved in a molar ratio of 0.5~1.5:2~4 to obtain a lactic acid-betaine eutectic solvent. Astragalus powder is mixed with the lactic acid-betaine eutectic solvent in a mass-volume ratio of 0.5~1.5g:5~15mL and then extracted by shaking in a water bath to obtain the extract. S2. After centrifuging the extract, collect the supernatant containing astragaloside A, remove excess lactic acid-betaine eutectic solvent, and the residue obtained is the astragaloside A extract.

2. The method for extracting astragaloside A according to claim 1, characterized in that, The dissolution conditions described in S1 are: 70-90°C water bath with magnetic stirring for 25-35 minutes.

3. The method for extracting astragaloside A according to claim 1, characterized in that, The water bath oscillation temperature described in S1 is 55~65℃.

4. The method for extracting astragaloside A according to claim 1, characterized in that, The oscillation speed is 110~130 rpm / min.

5. The method for extracting astragaloside A according to claim 1, characterized in that, The extraction time is 1 to 3 hours.

6. The method for extracting astragaloside A according to claim 1, characterized in that, The centrifugation conditions are 3000~5000 rpm for 10~20 min.

7. The method for extracting astragaloside A according to claim 1, characterized in that, The conditions for removing excess lactic acid-betaine eutectic solvent are 40~60℃ and 0.05~0.15MPa vacuum distillation.

8. The method for extracting astragaloside A according to claim 1, characterized in that, The viscosity of the lactic acid-betaine eutectic solvent is 400~600 mPa·s.

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