CN114617913A - Method for extracting, separating and purifying total saponins of astragalus stems and leaves - Google Patents
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Abstract
The invention discloses an extraction, separation and purification method of total saponins in stems and leaves of astragalus, which comprises the following steps: pulverizing stems and leaves of radix astragali, sieving with 60 mesh sieve, adding 60% ethanol at a material-to-liquid ratio of 1:25(g/mL), ultrasonic extracting at 57 deg.C for 61min, extracting with 1 volume of water saturated n-butanol for 2 times, and concentrating under reduced pressure to dry to obtain total saponin extract of stems and leaves of radix astragali; purifying the total saponin extract solution by AB-8 macroporous resin, and optimizing the optimal static adsorption conditions as follows: the adsorption time is 5h, the ratio of the resin mass to the sample liquid is 1:15, and the adsorption rate is 50.9 percent; optimal static desorption conditions: 85% absolute ethyl alcohol, the desorption time is 4h, the ratio of the resin mass to the sample liquid is 1:20, and the desorption rate is 56.6%; the purity of the astragalus stem and leaf saponin purified by the AB-8 macroporous resin reaches over 96 percent, and the antioxidant activity is enhanced by 110 to 120 percent compared with that before purification; the method simplifies the extraction and purification steps and saves the production cost; the method is simple to operate, easy to repeat and suitable for standard industrial production.
Description
Technical Field
The invention relates to the technical field of total saponins of astragalus stems and leaves, in particular to a method for extracting, separating and purifying total saponins of astragalus stems and leaves.
Background
Astragalus, also known as astragalus membranaceus, is one of the genuine medicinal materials in Shanxi, is the dried root of Astragalus mongholicus (Fisch.) bge or Astragalus membranaceus (Fisch.) bge, which is a traditional Chinese medicinal material with wide application.
Research shows that the chemical components of the astragalus root mainly comprise astragalus root total saponin, astragalus polysaccharide, flavonoid compounds and the like. The total saponins of astragalus have the functions of resisting inflammation, aging and virus, resisting hepatic fibrosis, easing pain, regulating immunity, regulating cardiovascular system and the like, and an important index commonly used for evaluating the quality of astragalus is the content of the total saponins of astragalus.
The chemical components contained in the stems and leaves of the astragalus are similar to those of the roots of the astragalus, and mainly comprise three substances of saponin, flavone and polysaccharide, wherein the saponin of the stems and leaves of the astragalus has the effects of regulating blood sugar in vivo, enhancing the immunity of the organism, promoting the growth and improving the oxidation resistance of the organism, and the release amount of the effective components of the traditional Chinese medicine directly influences the exertion of the medicine effect of the traditional Chinese medicine; according to the variety of the traditional Chinese medicine and the different properties of the effective active ingredients, a proper extraction process is selected to improve the extraction rate of the effective ingredients and improve the curative effect of the traditional Chinese medicine.
However, the traditional water decoction method, reflux method, ultrahigh pressure extraction method, enzyme method and microwave-assisted method all have certain disadvantages, so the extraction method of the total saponins in the stem and leaf of the astragalus is particularly important.
Disclosure of Invention
The invention aims to provide a method for extracting, separating and purifying total saponins from stems and leaves of astragalus to solve the problems in the background art.
In order to achieve the purpose, the invention provides the following technical scheme: a method for extracting, separating and purifying total saponins of stems and leaves of astragalus comprises the following steps:
s1: the method comprises the following steps of ultrasonically extracting ethanol crude extracts of astragalus stems and leaves:
s1: the method comprises the following steps of ultrasonically extracting ethanol crude extracts of astragalus stems and leaves:
1) putting the dried astragalus stems and leaves into a grinder, grinding into powder, sieving with a 60-mesh sieve, and weighing sieved astragalus stem and leaf powder;
2) adding ethanol solution with certain concentration according to a certain liquid-material ratio, performing ultrasonic extraction at a specified temperature for a specified time, performing suction filtration, and performing rotary evaporation to volatilize ethanol to obtain ethanol crude extract of astragalus stem and leaf;
s2: the method comprises the following steps of:
1) fully re-dissolving the ethanol crude extract of the astragalus stem and leaf with distilled water, then extracting for 2 times with water saturated n-butanol with the volume of 1 time, combining n-butanol phases, and performing rotary evaporation to dryness to obtain total saponin extracts of the astragalus stem and leaf;
2) metering the volume of the total saponin extract of the stem and leaf of the astragalus root in a volumetric flask by using methanol to obtain a sample solution of the total saponin extract of the stem and leaf of the astragalus root;
s3: purifying by macroporous resin, comprising the following specific steps:
1) soaking the resin in 95% ethanol for 24h, and fully swelling the resin to recover the pore structure of the resin;
2) continuously washing with 95% ethanol until turbidity does not occur when 1 part of effluent is added with 3 parts of pure water, and washing with purified water until no alcohol smell exists for later use;
3) pouring the sample liquid into resin, wherein the mass of the resin and the amount of the sample liquid are 1:15, the adsorption time is 5h, and the adsorption pH is 7;
4) pouring the desorption solution into the resin after adsorption, wherein the mass of the resin and the dosage of the desorption solution are 1:20, and the desorption time is 4 hours to obtain eluent;
s4: and drying the collected eluent to obtain a finished product.
In order to improve the extraction amount of the total saponins in the stems and leaves of the astragalus membranaceus, step S1 adopts a single-factor combined response surface method to optimize the extraction process, and the optimal ethanol concentration is determined to be 60%, the material-liquid ratio is 1:25(g/ml), the extraction time is 61min, and the extraction temperature is 57 ℃.
In order to improve the purity of the total saponins in stem and leaf of astragalus and improve the yield, in step S2, the extraction times of water saturated n-butanol is 2 times, and the extraction time of a single time is more than 20 min.
The total saponin extraction amount of the stem and leaf of the astragalus mongholicus is measured by adopting a vanillin-perchloric acid method, astragaloside is used as a reference substance, the absorbance is measured to be 0.6669 under the wavelength of 560nm, the standard curve is substituted to obtain the total saponin concentration (mu g/mL) of the extracting solution, and then the calculation is carried out according to the following formula:
the total saponin extraction amount (mg/g) of the astragalus stem leaves is (the total saponin concentration of the extracting solution is multiplied by the total volume of the measuring solution multiplied by the total volume of the extracting solution)/(the sampling volume of the extracting solution multiplied by the mass of the astragalus stem leaves multiplied by 1000);
wherein the total volume of the determination solution is 6mL, the total volume of the extracting solution is 50mL, the sampling volume of the extracting solution is 0.1mL, and the mass of the stem and leaf of the astragalus is 10 g.
In order to further improve the purity of the total saponins in the stems and leaves of the astragalus, the resin in the step S3 is AB-8 macroporous adsorption resin.
In addition, the desorption solution in step S3 was ethanol with a volume fraction of 85%.
In order to improve the stability of the product, in step S4, a vacuum reduced pressure drying method is adopted, and the eluent is concentrated to 40% -50% of the original volume by rotary evaporation in advance, wherein the vacuum reduced pressure drying conditions are as follows: the temperature is 50-60 ℃, and the pressure is 40-53 kPa.
In order to improve the stability of the product, in step S4, a freeze-drying method is adopted, wherein the freeze-drying conditions are as follows: the temperature is-10 to-50 ℃, and the pressure is 1.3 multiplied by 10-3～1.3×10-2kPa。
The in vitro antioxidant activity of the total saponins of astragalus stems and leaves prepared by the method for extracting, separating and purifying the total saponins of astragalus stems and leaves is compared, and the result shows that the cleaning capacity of the total saponins of astragalus stems and leaves to DPPH & and ABTS & lt + & gt after purification is 92% at most, 120% higher than that before purification, 88% higher than that before purification and 110% higher than that before purification by comparing the cleaning capacity of DPPH & and ABTS & lt + & gt.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention utilizes ethanol water solution to carry out ultrasonic extraction on the stem and leaf powder of the astragalus, optimizes the optimal extraction process by adopting a single-factor combined response surface method, determines that the ultrasonic extraction is optimal by using 60 percent low-concentration ethanol water solution, and improves the stability of the product.
2. The invention adopts organic solvent extraction to primarily separate saponin components, and adopts a vanillin-perchloric acid method to measure the total saponin extraction quantity of the astragalus stem leaves, so that the extraction frequency of water saturated n-butyl alcohol is 2 times, the single extraction time is not less than 20min, and the total saponin extraction quantity (calculated by astragaloside) of the astragalus stem leaves is 8.44mg/g, thereby improving the purity of the total saponin of the astragalus stem leaves and simultaneously improving the yield.
3. The invention uses macroporous adsorption resin to carry out adsorption and desorption, and respectively optimizes the adsorption and desorption processes by adopting a single-factor combined orthogonal experimental method, and finally obtains the optimal adsorption process which comprises the following steps: the ratio of the mass of the resin to the amount of the sample solution is 1:15, the adsorption time is 5h, the adsorption pH is 7, and the adsorption rate of the total saponins in the stem leaves of the astragalus membranaceus is 50.9 percent; the optimal desorption process is as follows: the volume fraction of ethanol in the desorption solution is 85 percent, the desorption time is 4 hours, the ratio of the mass of the resin to the dosage of the desorption solution is 1:20, and the desorption rate of the total saponins in the astragalus stems and leaves is 56.6 percent.
4. The invention properly concentrates the eluent and then carries out vacuum reduced pressure drying to obtain a finished product, the purity of the obtained astragalus stem and leaf saponin reaches over 96 percent, and the in vitro antioxidant activity of the total saponin of the astragalus stem and leaf before and after the purification by macroporous resin is compared, and the antioxidant activity is enhanced by 110 to 120 percent.
5. The method is simple to operate, easy to control, environment-friendly and suitable for realizing standard industrial production.
Drawings
FIG. 1 is a standard curve of astragaloside IV;
FIG. 2 is a curve showing the variation of total saponin content of astragalus stem and leaf with dynamic elution volume;
FIG. 3 shows the in vitro antioxidant activity of total saponins from stem and leaf of Astragalus before purification;
FIG. 4 shows the in vitro antioxidant activity of total saponins from stem and leaf of Astragalus after purification.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The first embodiment is as follows:
according to the figures 1-4, the method for extracting, separating and purifying the total saponins from the stems and leaves of the astragalus comprises the following steps:
s1: the method comprises the following steps of ultrasonically extracting ethanol crude extracts of astragalus stems and leaves:
1) putting the dried astragalus stems and leaves into a grinder, grinding into powder, sieving with a 60-mesh sieve, and weighing 10g of sieved astragalus stem and leaf powder;
2) adding 60% ethanol solution with liquid-material ratio of 1:25mL/g, dissolving completely, ultrasonic extracting at 57 deg.C for 61min, suction filtering, rotary evaporating to remove ethanol, adding 20mL distilled water, and standing to obtain ethanol crude extract of stem and leaf of radix astragali.
S2: the method comprises the following steps of:
1) extracting with 20mL of prepared water-saturated n-butanol twice for 20min each time, discarding water solution, mixing filtrates, and rotary evaporating to dry;
2) and (4) diluting the solution to a 50mL volumetric flask with methanol to obtain a sample solution of the total saponins in the stem and leaf of the astragalus.
Wherein, astragaloside IV is used as a reference substance, and the content of total saponins is determined by adopting a vanillin-perchloric acid method, which comprises the following steps: accurately weighing 5.000mg of astragaloside IV reference substance, dissolving in 10mL volumetric flask with methanol, preparing into standard solution, and numbering test tubes with stoppers as 1, 2, 3, 4, 5, and 6. Sucking 0.2mL, 0.3 mL, 0.4 mL, 0.5mL, 0.6 mL and 0.7mL of standard solution, volatilizing in water bath, adding 0.2mL of 5% vanillin-glacial acetic acid solution and 0.8mL of perchloric acid solution respectively, shaking uniformly, developing in a 70 ℃ water bath kettle, taking out after 20min, putting into ice water, cooling for 5min, taking out, adding 5mL of glacial acetic acid, and mixing uniformly. The absorbance was measured at 560nm using the same method as a methanol blank,the mass of the control solution was plotted on the abscissa and the absorbance A on the ordinate to obtain a regression equation Y of 0.00239X-0.0057, R20.9976, as shown in fig. 1.
Sucking 0.1-0.2 mL of total saponins in stems and leaves of astragalus membranaceus, volatilizing in a water bath, adding 0.8mL of perchloric acid solution and 0.2mL of 5% vanillin-glacial acetic acid solution, shaking uniformly, adding the mixture into a water bath kettle at 70 ℃ for color development, taking out after 20min, placing the mixture into ice water for cooling for 5min, taking out, adding 5mL glacial acetic acid, mixing uniformly, measuring the absorbance at 560nm, and repeating the measurement for three times to obtain the average absorbance value A. And respectively calculating the quality of the saponin according to the corresponding regression equation.
The total saponin extraction amount (mg/g) is (total saponin concentration of the extracting solution is multiplied by total volume of the measuring solution multiplied by total volume of the extracting solution)/(sampling volume of the extracting solution multiplied by stem and leaf mass of astragalus root multiplied by 1000), wherein the total volume of the measuring solution is 6mL, the total volume of the extracting solution is 50mL, the sampling volume of the extracting solution is 0.1-0.2 mL, and the mass of stem and leaf of astragalus root is 10 g.
And performing 3 parallel verification experiments to obtain an average absorbance value of 0.6669, and obtaining an average value of 8.44mg/g of the total saponins in the stems and leaves of the astragalus according to the formula.
S3: purifying by macroporous resin, comprising the following specific steps:
1) soaking AB-8 resin in 95% ethanol for 24 hr, and swelling the resin to restore the pore structure;
comparing the three macroporous resins D101, D201 and AB-8, and finally determining the macroporous resin as the optimal macroporous adsorption resin suitable for purifying the total saponins of astragalus stems and leaves.
2) Continuously washing with 95% ethanol until turbidity does not appear when adding 3 parts of pure water into 1 part of effluent, and washing with purified water until no alcohol smell exists for later use;
3) metering the volume of the total saponin extract of the stem and leaf of the astragalus root in a volumetric flask by using methanol to obtain a sample solution of the total saponin extract of the stem and leaf of the astragalus root;
4) pouring the sample liquid into resin, wherein the mass of the resin and the amount of the sample liquid are 1:15, the adsorption time is 5h, and the adsorption pH is 7;
5) and pouring the desorption solution into the resin after adsorption, wherein the mass of the resin and the dosage of the desorption solution are 1:20, and the desorption time is 4h, so as to obtain the eluent.
The method for measuring the adsorption rate and the resolution ratio comprises the following steps:
sucking 0.1mL of the extractive solution of the stem and leaf of radix astragali, adsorbing with resin, and desorbing, respectively, placing in a test tube with a plug, volatilizing in water bath, adding 0.2mL of 5% vanillin-glacial acetic acid solution and 0.8mL of perchloric acid, heating in a constant temperature water bath at 70 deg.C for 20min, cooling with flowing water for 5min, adding 5mL of glacial acetic acid, shaking, and developing. And (3) measuring the absorbance at 560nm by taking the corresponding reagent as a blank, and calculating the adsorption rate and the desorption rate of the resin on the total saponin. The calculation formula is as follows: adsorption rate [ [ (C)0-C1)/C0]X 100%; desorption rate [ (V)2×C2)/V1×(C0-C1)]×100％。
In the formula C0The mass concentration (mg/mL) of total saponin in the initial astragalus stem and leaf extracting solution is adopted; c1The mass concentration (mg/mL) of total saponins in the solution after resin adsorption; c2The mass concentration (mg/mL) of the total saponins in the astragalus stems in the desorption solution; v1Is the volume (mL) of the stem and leaf sample solution of the astragalus; v2Volume of stripping solution (mL).
The adsorption and desorption processes are respectively optimized by combining a single factor with an orthogonal experimental method. The final optimal adsorption process is as follows: the resin mass is that the dosage of the sample liquid is 1:15, the adsorption time is 5h, the adsorption pH is 7, and the adsorption rate of the total saponins in the astragalus stems and leaves is 50.9 percent; the optimal desorption process comprises the following steps: the volume fraction of the stripping solution ethanol is 85 percent, the stripping time is 4h, the resin mass is 1:20, and the stripping rate of the total saponins in the astragalus stems and leaves is 56.6 percent.
Dynamic analysis: loading a certain amount of pretreated AB-8 macroporous adsorbent resin into a column by a wet method, infiltrating the macroporous resin with saponin extract, fully and uniformly stirring the AB-8 macroporous resin in the extract by a glass rod, and pouring the resin and sample liquid into a glass chromatographic column at one time (preventing bubbles from being generated between the resins).
After sample adsorption for 5h, eluting with 85% absolute ethyl alcohol at a flow rate of 0.5mL/min, collecting the effluent liquid step by step, collecting 80 tubes in total, combining each 10 tubes into one component, measuring the concentration of total saponin in each component, and then drawing a dynamic adsorption-desorption curve (as shown in figure 2) by taking the volume of the eluate as an abscissa and the concentration of total saponin in the effluent liquid of each component as an ordinate.
The AB-8 resin has good elution performance on total saponins, and elution peaks are relatively concentrated; when the eluent is in the 2 nd to 5 th components (namely, the elution volume is 20-50 mL), the saponin content in the eluent is higher, and the eluent of the 2 nd to 5 th components is mainly collected in the subsequent purification process for subsequent antioxidant determination.
In order to verify the purity of the product, the in-vitro antioxidant activity of the total saponins of astragalus stems and leaves before and after purification by macroporous resin is compared, and the result of comparison of the removing capacity of DPPH & and ABTS & lt + & gt shows that the removing capacity of the total saponins of astragalus stems and leaves after purification to DPPH & is 92% at most, is improved by 120% compared with that before purification, and is 88% compared with that before purification, and is improved by 110% compared with that before purification (as shown in figures 3 and 4).
S4: and drying the collected eluent to obtain a finished product.
In order to improve the stability of the product, in step S4, the product is dried by vacuum reduced pressure drying, the eluent is concentrated to 40% -50% of the original volume by rotary evaporation in advance, and the vacuum reduced pressure drying conditions are as follows: the temperature is 50-60 ℃, and the pressure is 40-53 kPa.
Example two:
other conditions are unchanged, in step S4, a freeze-drying method is adopted to realize product drying, the eluent is concentrated to 40-50% of the original volume by rotary evaporation in advance, and the adopted freeze-drying conditions are as follows: the temperature is-10 to-50 ℃, and the pressure is 1.3 multiplied by 10-3～1.3×10-2kPa。
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (9)
1. A method for extracting, separating and purifying total saponins from stems and leaves of astragalus is characterized by comprising the following steps: the method comprises the following steps:
s1: the method comprises the following steps of ultrasonically extracting ethanol crude extracts of astragalus stems and leaves:
1) putting the dried astragalus stems and leaves into a grinder, grinding into powder, sieving with a 60-mesh sieve, and weighing sieved astragalus stem and leaf powder;
2) adding ethanol solution with certain concentration according to a certain liquid-material ratio, performing ultrasonic extraction at a specified temperature for a specified time, performing suction filtration, and performing rotary evaporation to volatilize ethanol to obtain ethanol crude extract of astragalus stem and leaf;
s2: the method comprises the following steps of:
1) fully re-dissolving the crude ethanol extract of the stem and leaf of the astragalus by using distilled water, then extracting for 2 times by using water saturated n-butyl alcohol with the volume of 1 time, combining n-butyl alcohol phases, and carrying out rotary evaporation to dryness to obtain total saponin extracts of the stem and leaf of the astragalus;
2) metering the volume of the total saponin extract of the stem and leaf of the astragalus root in a volumetric flask by using methanol to obtain a sample solution of the total saponin extract of the stem and leaf of the astragalus root;
s3: macroporous resin purification, which comprises the following steps:
1) soaking the resin in 95% ethanol for 24h, and fully swelling the resin to recover the pore structure of the resin;
2) continuously washing with 95% ethanol until turbidity does not occur when 1 part of effluent is added with 3 parts of pure water, and washing with purified water until no alcohol smell exists for later use;
3) pouring the sample liquid into resin, wherein the mass of the resin and the amount of the sample liquid are 1:15, the adsorption time is 5h, and the adsorption pH is 7;
4) pouring the desorption solution into the resin after adsorption, wherein the mass of the resin and the dosage of the desorption solution are 1:20, and the desorption time is 4 hours to obtain eluent;
s4: and drying the collected eluent to obtain a finished product.
2. The method for extracting, separating and purifying the total saponins of astragalus stems and leaves as claimed in claim 1, which is characterized in that: step S1 is to optimize the extraction process by combining single factor with response surface method, to determine the optimal ethanol concentration of 60%, material-to-liquid ratio of 1:25(g/ml), extraction time of 61min, and extraction temperature of 57 deg.C.
3. The method for extracting, separating and purifying the total saponins of astragalus stems and leaves as claimed in claim 1, which is characterized in that: in step S2, the extraction times of water saturated n-butanol is 2 times, and the single extraction time is more than 20 min.
4. The method for extracting, separating and purifying total saponins of stem and leaf of astragalus as claimed in claim 3, which is characterized in that: the total saponin extraction amount of the stem and leaf of the astragalus mongholicus is measured by adopting a vanillin-perchloric acid method, astragaloside is used as a reference substance, the absorbance is measured to be 0.6669 under the wavelength of 560nm, the standard curve is substituted to obtain the total saponin concentration (mu g/mL) of the extracting solution, and then the calculation is carried out according to the following formula:
the total saponin extraction amount (mg/g) of the astragalus stem and leaf is (the total saponin concentration of the extracting solution is multiplied by the total volume of the measuring solution multiplied by the total volume of the extracting solution)/(the sampling volume of the extracting solution multiplied by the mass of the astragalus stem and leaf multiplied by 1000).
5. The method for extracting, separating and purifying the total saponins of astragalus stems and leaves as claimed in claim 1, which is characterized in that: the resin in the step S3 is AB-8 macroporous adsorption resin.
6. The method for extracting, separating and purifying the total saponins of astragalus stems and leaves as claimed in claim 1, which is characterized in that: the desorption solution in step S3 was ethanol with a volume fraction of 85%.
7. The method for extracting, separating and purifying the total saponins of astragalus stems and leaves as claimed in claim 1, which is characterized in that: in step S4, a vacuum reduced pressure drying method is adopted, the eluent is concentrated to 40% -50% of the original volume by rotary evaporation in advance, and the adopted vacuum reduced pressure drying conditions are as follows: the temperature is 50-60 ℃, and the pressure is 40-53 kPa.
8. The method for extracting, separating and purifying total saponins of stem and leaf of astragalus according to claim 1The method is characterized in that: in step S4, a freeze-drying method is employed, the freeze-drying conditions being: the temperature is-10 to-50 ℃, and the pressure is 1.3 multiplied by 10-3～1.3×10- 2kPa。
9. An application of the total saponins of astragalus stems and leaves prepared by the method for extracting, separating and purifying the total saponins of astragalus stems and leaves as claimed in claim 1 in-vitro antioxidant activity is characterized in that: the in-vitro antioxidant activity of the total saponins of astragalus stems and leaves before and after purification by macroporous resin is compared, and the result of comparison of the removal capacity of DPPH & ABTS +. shows that the removal capacity of the total saponins of astragalus stems and leaves after purification to DPPH & is 92% at most, the removal capacity to ABTS +. is 88% at most, and the removal capacity to ABTS +. is 110% at most.
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