CN106990153A - A kind of method for differentiating astragalus mongolicus and Astragalus membranacus - Google Patents

Abstract

The invention relates to a method for identifying Astragalus mongolica and Astragalus membranaceus. The purpose of the invention is to solve the technical problems that the existing identification methods of Astragalus mongolica and Astragalus membranaceus have low specificity and are difficult to identify the species of Astragalus membranaceus; the technical solution of the present invention It is: drying and crushing the medicinal material of Astragalus membranaceus; extracting astragalus polysaccharides by water extraction and alcohol precipitation; preparing partial acid hydrolyzate of astragalus polysaccharide; derivatization of hydrolyzate; electrophoresis analysis of astragalus polysaccharide hydrolyzate; Calculate the content of trisaccharides and disaccharides in Astragalus polysaccharides, and identify Astragalus mongolica and Astragalus membranaceus according to the following criteria: when the disaccharide content is less than 1.0 mg/mL, and the disaccharide content/trisaccharide content is less than 0.3, it is Astragalus mongolica; When disaccharide content > 1.0mg/mL, and disaccharide content/trisaccharide content > 0.3, it is Astragalus membranaceus; the invention not only has simple operation and low experiment cost, but also is formed after partial acid hydrolysis of astragalus polysaccharides of different species The sugar components are significantly different, which can be used to identify the species of Astragalus membranaceus.

Description

A method for distinguishing Astragalus mongolica and Astragalus membranaceus

technical field

The invention relates to a method for identifying Astragalus mongolica and Astragalus membranaceus, which specifically belongs to fluorescence-assisted gel electrophoresis technology. The method for identifying Astragalus mongolica and Astragalus membranaceus is based on the content of differential sugar fragments after partial acid hydrolysis of astragalus polysaccharides. .

Background technique

Astragalus, known as Astragalus in ancient times, has the reputation of "the strength of tonic". It is recorded in the "Chinese Pharmacopoeia" in the past editions. It is the dried root of the bean plant Astragalus mongolica or Astragalus membranaceus. , curb sores and promote granulation and other effects, there is a saying of "ten medicines and nine qi", and has been favored by traditional Chinese medicine. And there are more than 200 kinds of Chinese patent medicines that use Astragalus as raw materials. The domestic market has a great demand for Astragalus, of which about 50% are used for the production of Astragalus decoction pieces, and nearly 50% are used for Chinese patent medicines, extracts and preparations.

Astragalus mongolica and Astragalus membranaceus have different medicinal effects due to differences in chemical components. Although there are obvious differences in the morphological and biological characteristics of the aboveground parts of the plants, the medicinal parts of Astragalus root are similar in appearance and difficult to detect with the naked eye. the difference. Therefore, in recent years, two kinds of Astragalus have appeared in the market of medicinal materials, which has disrupted the market of traditional Chinese medicinal materials.

Clinical studies have shown that sugar components are the main substances that Astragalus plays an immune-regulating role. It has a good research prospect and significance to use sugar components as the standard of quality control and quality evaluation of Radix Astragali. Scholars at home and abroad have also tried to compare different species of Astragalus membranaceus with the total polysaccharide content. However, the measured indicators lack the specific characteristics of Astragalus membranaceus species, so it is difficult to be used for identification of Astragalus membranaceus species.

Contents of the invention

The purpose of the present invention is to solve the technical problem that the existing identification method of Astragalus mongolica and Astragalus membranaceus has low specificity and is difficult to identify the species of Astragalus membranaceus, and provides a method for identifying Astragalus mongolica and Astragalus membranaceus.

In order to solve the above-mentioned technical problems, the technical scheme adopted in the present invention is: a kind of method for distinguishing Astragalus mongolica and Astragalus membranaceus, comprising the following steps:

1) Raw material pretreatment: crush the dried Astragalus membranaceus into powder and pass through a 100-mesh sieve to obtain Astragalus membranaceus fine powder, which is set aside;

2) Extraction of crude polysaccharides: take the above-mentioned astragalus fine powder and add water, reflux and extract at 100°C for 1 hour, cool down to room temperature, and centrifuge to obtain the supernatant; add water to the residue and repeat the extraction once, take the supernatant, and combine the supernatant; Concentrate, adjust the combined supernatant to 80% alcohol content with 95% ethanol, centrifuge at 3000r/min for 10min, combine the precipitates, freeze-dry polysaccharide coarse powder;

3) Purification of polysaccharide: dissolving the coarse polysaccharide powder obtained in step 2) in water to obtain a coarse polysaccharide powder solution, the mass ratio of the coarse polysaccharide powder to water is 1:200, and the concentration of 10.6% is added to the coarse polysaccharide powder solution Potassium ferrocyanide solution and concentration are the zinc acetate solution of 21.9%, and described concentration is that the add-on of the potassium ferrocyanide solution of 10.6% and the zinc acetate solution of 21.9% are polysaccharide coarse powder solution volume 10%, shake and let stand for 30 minutes, centrifuge to obtain purified polysaccharide, freeze-dry, and set aside;

4) Polysaccharide acid hydrolysis: Take 10 mg of the purified polysaccharide obtained in step 3), add 500 μL of 0.5 mol/L trifluoroacetic acid, mix well, then place the mixture at 90°C for 1 hour, after the reaction, dry it with a nitrogen blowing dryer Polysaccharide acid hydrolyzate, then add an appropriate amount of methanol to clean the hydrolyzate, and continue to blow dry with nitrogen;

5) Derivatization of polysaccharide acid hydrolyzate, trisaccharide standard and disaccharide standard: Take 6 mg of trisaccharide standard, 2 mg of disaccharide standard and the polysaccharide acid hydrolyzate obtained in step 4), add 200 μL of NaCNBH3 solution respectively , and then add 200 μL of ANTS derivatization reagent, vortex, and mix well; after 15 hours of constant temperature reaction at 37 ° C, the three products are dried with nitrogen, and dissolved in 1 mL of 6mol/L urea solution to prepare trisaccharide standard products, disaccharides Derivatized samples of sugar standard and polysaccharide acid hydrolyzate, among which the derivatized samples of trisaccharide standard and disaccharide standard are diluted with 6mol/L urea solution to form derivatized samples with no less than 6 concentration gradients;

6) Electrophoresis analysis of derivatized samples: take the polysaccharide acid hydrolyzate derivatized samples, trisaccharide standard derivatized samples and disaccharide standard derivatized samples in step 5), and use vertical plate gel electrophoresis to separate each derivatized sample, The separation gel is a polyacrylamide gel with a concentration of 30%, the stacking gel is a polyacrylamide gel with a concentration of 8%, and the electrophoresis buffer is Tris-boric with a pH of 8.0-9.0 and a concentration of 0.1-0.2mol/L. The sample volume is 1-3 μL, and the gel is imaged under UV300nm conditions to obtain the electrophoresis of polysaccharide acid hydrolyzate derivatized samples, trisaccharide standard derivatized samples and disaccharide standard derivatized samples;

7) Draw the standard curve of the trisaccharide standard and the disaccharide standard: import the electropherograms of the polysaccharide acid hydrolyzate derivatized sample, the trisaccharide standard derivatized sample and the disaccharide standard derivatized sample into Quantity One software, after processing Afterwards, obtain the optical density value of polysaccharide acid hydrolyzate and trisaccharide standard substance and disaccharide standard substance, take the optical density value of trisaccharide standard substance and disaccharide standard substance as ordinate, concentration as abscissa, draw standard curve;

8) Identification of Astragalus mongolica and Astragalus membranaceus: Bring the optical density value of the polysaccharide acid hydrolyzate into the standard curve in step 7), calculate the content of trisaccharides and disaccharides in Astragalus polysaccharide, and identify Astragalus mongolica and Astragalus membranaceus according to the following criteria Astragalus membranaceus:

When the disaccharide content is less than 1.0mg/mL, and the disaccharide content/trisaccharide content is less than 0.3, it is Astragalus mongolica;

When the disaccharide content>1.0mg/mL, and the disaccharide content/trisaccharide content>0.3, it is Astragalus membranaceus.

Further, the mass ratio of Astragalus fine powder to water in step 2) is 1:100.

Further, the mass ratio of residue to water in step 2) is 1:80.

Further, the preparation solvent of the separating gel and the stacking gel in step 6) is a Tris-boric buffer solution with a pH of 8.0-9.0 and a concentration of 0.1-0.2 mol/L.

Further, the electrophoretic separation process in step 6) is carried out in two steps. Firstly, a separation voltage of 60-80V is used for electrophoretic separation for 40-60 min, and then a separation voltage of 100-200V is used for electrophoretic separation for 100-120 min.

The beneficial effects of the present invention are:

1) The present invention partially acid-hydrolyzes the polysaccharides in Astragalus membranaceus, and uses fluorescence-assisted gel electrophoresis analysis to find out the difference in the sugar components of Astragalus mongolica and Astragalus membranaceus, which is used to identify the species of Astragalus membranaceus. This method is not only easy to operate and low in experiment cost, but also the sugar components formed by partial acid hydrolysis of different species of Astragalus polysaccharides are significantly different, which can be used to identify the species of Astragalus medicinal materials.

2) The fluorescence-assisted gel electrophoresis (PACE) technology used in the present invention is a very convenient polysaccharide hydrolyzate separation technology, which has the characteristics of high resolution, good repeatability, high stability, and simultaneous analysis of multiple samples; Since the carbohydrate compound does not have an ultraviolet or fluorescent group, the present invention uses the derivatization reagent 8-aminonaphthalene-1,3,6-sodium trisulfonate (ANTS) to derivatize the polysaccharide hydrolyzate before performing PACE analysis , so that the polysaccharide hydrolyzate carries ultraviolet groups and fluorescent groups, so that the detection sensitivity is greatly improved.

Description of drawings

Fig. 1 is the polyacrylamide gel electrophoresis figure of partial acid hydrolyzate of polysaccharides of astragalus of different species;

Fig. 2 is the fingerprint of different species of astragalus polysaccharide partial acid hydrolyzate;

Fig. 3 is the T test analysis diagram of differential sugar fragments;

Fig. 4 is the PLS-DA figure of the partial acid hydrolyzate of astragalus polysaccharide of different species;

Figure 5 is a verification diagram of the PLS-DA analysis model;

Figure 6 is a loading diagram of differential sugar fragments;

Figure 7 is a graph showing the number of differential sugar fragments of partial acid hydrolyzates of astragalus polysaccharides of different species;

Fig. 8 is the standard curve figure of disaccharide standard substance;

Figure 9 is a standard curve diagram of a trisaccharide standard.

detailed description

The present invention will be further described below in conjunction with drawings and embodiments.

Example 1

A method for identifying Astragalus mongolica and Astragalus membranaceus in the present embodiment comprises the following steps:

1) Raw material pretreatment: crush the dried Astragalus membranaceus into powder and pass through a 100-mesh sieve to obtain Astragalus membranaceus fine powder, which is set aside;

2) Extraction of crude polysaccharides: take about 5 g of the above-mentioned astragalus fine powder, accurately weigh it, place it in a 1000 mL round bottom flask, add 500 mL of water, reflux and extract at 100 ° C for 1 h, cool down to room temperature, and centrifuge to take the supernatant; Add 400mL of water, repeat the extraction once, take the supernatant, combine the supernatant; concentrate, adjust the combined supernatant to 80% alcohol content with 95% ethanol, centrifuge at 3000r/min for 10min, combine the precipitate, and freeze-dry much sugar meal;

3) Purification of polysaccharide: the polysaccharide coarse powder obtained in step 2) is dissolved in water to obtain a polysaccharide coarse powder solution, the mass ratio of the polysaccharide coarse powder to water is 1:200, and a concentration of 10.6% of Potassium ferrocyanide solution and concentration are the zinc acetate solution of 21.9%, and described concentration is that the add-on of the potassium ferrocyanide solution of 10.6% and the zinc acetate solution of 21.9% are 10% of the polysaccharide coarse powder solution volume. %, shake and let stand for 30 minutes, centrifuge to obtain purified polysaccharide, lyophilize and set aside;

4) Polysaccharide acid hydrolysis: Take 10 mg of the purified polysaccharide obtained in step 3), add 500 μL of 0.5 mol/L trifluoroacetic acid, mix well, then place the mixture at 90°C for 1 hour, after the reaction, dry it with a nitrogen blowing dryer Polysaccharide acid hydrolyzate to remove the residual trifluoroacetic acid, then add an appropriate amount of methanol to clean the hydrolyzate, and continue to blow dry with nitrogen;

5) Derivatization of polysaccharide acid hydrolyzate, trisaccharide standard and disaccharide standard: Take 6 mg of trisaccharide standard, 2 mg of disaccharide standard and the polysaccharide acid hydrolyzate obtained in step 4), add 200 μL of NaCNBH3 solution respectively , and then add 200 μL of ANTS derivatization reagent, vortex, and mix well; after 15 hours of constant temperature reaction at 37 ° C, the three products are dried with nitrogen, and dissolved in 1 mL of 6mol/L urea solution to prepare trisaccharide standard products, disaccharides Derivatized samples of sugar standard and polysaccharide acid hydrolyzate, among which the derivatized samples of trisaccharide standard and disaccharide standard are diluted with 6mol/L urea solution to form derivatized samples with no less than 6 concentration gradients;

6) Electrophoresis analysis of derivatized samples: take the polysaccharide acid hydrolyzate derivatized samples, trisaccharide standard derivatized samples and disaccharide standard derivatized samples in step 5), and use vertical plate gel electrophoresis to separate each derivatized sample The separation gel is a polyacrylamide gel with a concentration of 30%, and the stacking gel is a polyacrylamide gel with a concentration of 8%, and the preparation solvents of the separation gel and the stacking gel are pH8.2, concentration 0.1mol/L Tris-boric buffer; the electrophoresis buffer is Tris-boric with pH 8.2 and concentration 0.1mol/L, and the loading volume is 1 μL; firstly, the separation voltage of 60V is used for electrophoresis separation for 60min, and then the separation voltage of 200V is used for electrophoresis separation for 120min. ; The gel was imaged under UV300nm conditions to obtain the electrophoresis of the polysaccharide acid hydrolyzate derivatized sample, the trisaccharide standard derivatized sample and the disaccharide standard derivatized sample;

7) Draw the standard curve of the trisaccharide standard and the disaccharide standard: import the electropherograms of the polysaccharide acid hydrolyzate derivatized sample, the trisaccharide standard derivatized sample and the disaccharide standard derivatized sample into Quantity One software, after processing Afterwards, obtain the optical density value of polysaccharide acid hydrolyzate and trisaccharide standard substance and disaccharide standard substance, take the optical density value of trisaccharide standard substance and disaccharide standard substance as ordinate, concentration as abscissa, draw standard curve;

8) Identification of Astragalus mongolica and Astragalus membranaceus: Bring the optical density value of the polysaccharide acid hydrolyzate into the standard curve in step 7), calculate the content of trisaccharides and disaccharides in Astragalus polysaccharide, and identify Astragalus mongolica and Astragalus membranaceus according to the following criteria Astragalus membranaceus:

When the disaccharide content is less than 1.0mg/mL, and the disaccharide content/trisaccharide content is less than 0.3, it is Astragalus mongolica;

When the disaccharide content>1.0mg/mL, and the disaccharide content/trisaccharide content>0.3, it is Astragalus membranaceus.

In this example, the calculated disaccharide content = 0.945 mg/mL, disaccharide content/trisaccharide content = 0.289, and the identification result is Astragalus mongolica.

Example 2

A method for identifying Astragalus mongolica and Astragalus membranaceus in the present embodiment comprises the following steps:

1) Raw material pretreatment: crush the dried Astragalus membranaceus into powder and pass through a 100-mesh sieve to obtain Astragalus membranaceus fine powder, which is set aside;

2) Extraction of crude polysaccharides: take about 5 g of the above-mentioned astragalus fine powder, accurately weigh it, place it in a 1000 mL round bottom flask, add 500 mL of water, reflux and extract at 100 ° C for 1 h, cool down to room temperature, and centrifuge to take the supernatant; Add 400mL of water, repeat the extraction once, take the supernatant, combine the supernatant; concentrate, adjust the combined supernatant to 80% alcohol content with 95% ethanol, centrifuge at 3000r/min for 10min, combine the precipitate, and freeze-dry much sugar meal;

3) Purification of polysaccharide: the polysaccharide coarse powder obtained in step 2) is dissolved in water to obtain a polysaccharide coarse powder solution, the mass ratio of the polysaccharide coarse powder to water is 1:200, and a concentration of 10.6% of Potassium ferrocyanide solution and concentration are the zinc acetate solution of 21.9%, and described concentration is that the add-on of the potassium ferrocyanide solution of 10.6% and the zinc acetate solution of 21.9% are 10% of the polysaccharide coarse powder solution volume. %, shake and let stand for 30 minutes, centrifuge to obtain purified polysaccharide, lyophilize and set aside;

4) Polysaccharide acid hydrolysis: Take 10 mg of the purified polysaccharide obtained in step 3), add 500 μL of 0.5 mol/L trifluoroacetic acid, mix well, then place the mixture at 90°C for 1 hour, after the reaction, dry it with a nitrogen blowing dryer Polysaccharide acid hydrolyzate to remove the residual trifluoroacetic acid, then add an appropriate amount of methanol to clean the hydrolyzate, and continue to blow dry with nitrogen;

5) Derivatization of polysaccharide acid hydrolyzate, trisaccharide standard and disaccharide standard: Take 6 mg of trisaccharide standard, 2 mg of disaccharide standard and the polysaccharide acid hydrolyzate obtained in step 4), add 200 μL of NaCNBH3 solution respectively , and then add 200 μL of ANTS derivatization reagent, vortex, and mix well; after 15 hours of constant temperature reaction at 37 ° C, the three products are dried with nitrogen, and dissolved in 1 mL of 6mol/L urea solution to prepare trisaccharide standard products, disaccharides Derivatized samples of sugar standard and polysaccharide acid hydrolyzate, among which the derivatized samples of trisaccharide standard and disaccharide standard are diluted with 6mol/L urea solution to form derivatized samples with no less than 6 concentration gradients;

6) Electrophoresis analysis of derivatized samples: take the polysaccharide acid hydrolyzate derivatized samples, trisaccharide standard derivatized samples and disaccharide standard derivatized samples in step 5), and use vertical plate gel electrophoresis to separate each derivatized sample The separation gel is a polyacrylamide gel with a concentration of 30%, and the stacking gel is a polyacrylamide gel with a concentration of 8%. The preparation solvents of the separation gel and the stacking gel are pH8.5, concentration 0.2mol/L Tris-boric buffer; the electrophoresis buffer is Tris-boric with a pH of 8.5 and a concentration of 0.2 mol/L, and the loading volume is 1 μL; firstly, a separation voltage of 70V is used for electrophoresis separation for 50 min, and then a separation voltage of 150 V is used for electrophoresis separation for 110 min. ; The gel was imaged under UV300nm conditions to obtain the electrophoresis of the polysaccharide acid hydrolyzate derivatized sample, the trisaccharide standard derivatized sample and the disaccharide standard derivatized sample;

7) Draw the standard curve of the trisaccharide standard and the disaccharide standard: import the electropherograms of the polysaccharide acid hydrolyzate derivatized sample, the trisaccharide standard derivatized sample and the disaccharide standard derivatized sample into Quantity One software, after processing Afterwards, obtain the optical density value of polysaccharide acid hydrolyzate and trisaccharide standard substance and disaccharide standard substance, take the optical density value of trisaccharide standard substance and disaccharide standard substance as ordinate, concentration as abscissa, draw standard curve;

8) Identification of Astragalus mongolica and Astragalus membranaceus: Bring the optical density value of the polysaccharide acid hydrolyzate into the standard curve in step 7), calculate the content of trisaccharides and disaccharides in Astragalus polysaccharide, and identify Astragalus mongolica and Astragalus membranaceus according to the following criteria Astragalus membranaceus:

When the disaccharide content is less than 1.0mg/mL, and the disaccharide content/trisaccharide content is less than 0.3, it is Astragalus mongolica;

When the disaccharide content>1.0mg/mL, and the disaccharide content/trisaccharide content>0.3, it is Astragalus membranaceus.

In this example, the calculated disaccharide content = 1.06 mg/mL, disaccharide content/trisaccharide content = 0.35, and the identification result is Astragalus membranaceus.

Example 3

A method for identifying Astragalus mongolica and Astragalus membranaceus in the present embodiment comprises the following steps:

1) Raw material pretreatment: crush the dried Astragalus membranaceus into powder and pass through a 100-mesh sieve to obtain Astragalus membranaceus fine powder, which is set aside;

2) Extraction of crude polysaccharides: take about 5 g of the above-mentioned astragalus fine powder, accurately weigh it, place it in a 1000 mL round bottom flask, add 500 mL of water, reflux and extract at 100 ° C for 1 h, cool down to room temperature, and centrifuge to take the supernatant; Add 400mL of water, repeat the extraction once, take the supernatant, combine the supernatant; concentrate, adjust the combined supernatant to 80% alcohol content with 95% ethanol, centrifuge at 3000r/min for 10min, combine the precipitate, and freeze-dry much sugar meal;

3) Purification of polysaccharide: the polysaccharide coarse powder obtained in step 2) is dissolved in water to obtain a polysaccharide coarse powder solution, the mass ratio of the polysaccharide coarse powder to water is 1:200, and a concentration of 10.6% of Potassium ferrocyanide solution and concentration are the zinc acetate solution of 21.9%, and described concentration is that the add-on of the potassium ferrocyanide solution of 10.6% and the zinc acetate solution of 21.9% are 10% of the polysaccharide coarse powder solution volume. %, shake and let stand for 30 minutes, centrifuge to obtain purified polysaccharide, lyophilize and set aside;

4) Polysaccharide acid hydrolysis: Take 10 mg of the purified polysaccharide obtained in step 3), add 500 μL of 0.5 mol/L trifluoroacetic acid, mix well, then place the mixture at 90°C for 1 hour, after the reaction, dry it with a nitrogen blowing dryer Polysaccharide acid hydrolyzate to remove the residual trifluoroacetic acid, then add an appropriate amount of methanol to clean the hydrolyzate, and continue to blow dry with nitrogen;

5) Derivatization of polysaccharide acid hydrolyzate, trisaccharide standard and disaccharide standard: Take 6 mg of trisaccharide standard, 2 mg of disaccharide standard and the polysaccharide acid hydrolyzate obtained in step 4), add 200 μL of NaCNBH3 solution respectively , and then add 200 μL of ANTS derivatization reagent, vortex, and mix well; after 15 hours of constant temperature reaction at 37 ° C, the three products are dried with nitrogen, and dissolved in 1 mL of 6mol/L urea solution to prepare trisaccharide standard products, disaccharides Derivatized samples of sugar standard and polysaccharide acid hydrolyzate, among which the derivatized samples of trisaccharide standard and disaccharide standard are diluted with 6mol/L urea solution to form derivatized samples with no less than 6 concentration gradients;

6) Electrophoresis analysis of derivatized samples: take the polysaccharide acid hydrolyzate derivatized samples, trisaccharide standard derivatized samples and disaccharide standard derivatized samples in step 5), and use vertical plate gel electrophoresis to separate each derivatized sample The separation gel is a polyacrylamide gel with a concentration of 30%, and the stacking gel is a polyacrylamide gel with a concentration of 8%. The preparation solvents of the separation gel and the stacking gel are pH9.0, concentration 0.15mol/L Tris-boric buffer; the electrophoresis buffer is Tris-boric with pH 9.0 and concentration 0.15mol/L, and the loading volume is 1μL; firstly, the separation voltage of 80V is used for electrophoresis separation for 40min, and then the separation voltage of 100V is used for electrophoresis separation for 100min. ; The gel was imaged under UV300nm conditions to obtain the electrophoresis of the polysaccharide acid hydrolyzate derivatized sample, the trisaccharide standard derivatized sample and the disaccharide standard derivatized sample;

7) Draw the standard curve of the trisaccharide standard and the disaccharide standard: import the electropherograms of the polysaccharide acid hydrolyzate derivatized sample, the trisaccharide standard derivatized sample and the disaccharide standard derivatized sample into Quantity One software, after processing Afterwards, obtain the optical density value of polysaccharide acid hydrolyzate and trisaccharide standard substance and disaccharide standard substance, take the optical density value of trisaccharide standard substance and disaccharide standard substance as ordinate, concentration as abscissa, draw standard curve;

8) Identification of Astragalus mongolica and Astragalus membranaceus: Bring the optical density value of the polysaccharide acid hydrolyzate into the standard curve in step 7), calculate the content of trisaccharides and disaccharides in Astragalus polysaccharide, and identify Astragalus mongolica and Astragalus membranaceus according to the following criteria Astragalus membranaceus:

When the disaccharide content is less than 1.0mg/mL, and the disaccharide content/trisaccharide content is less than 0.3, it is Astragalus mongolica;

When the disaccharide content>1.0mg/mL, and the disaccharide content/trisaccharide content>0.3, it is Astragalus membranaceus.

In this example, the calculated disaccharide content = 1.225 mg/mL, disaccharide content/trisaccharide content = 0.287, and the identification result is Astragalus mongolica.

The disaccharide standard and trisaccharide standard in the above examples are from China National Institute for the Control of Pharmaceutical and Biological Products, and the batch numbers are 100287-201102 and 100274, respectively.

The standard for identifying the species of Astragalus in the present invention is obtained through the following experiments.

Astragalus membranaceus used in the experiment was taken from Longxi, Gansu and Wendeng, Shandong, and Astragalus membranaceus was taken from Hunyuan, Shanxi and Heilongjiang, and 12 samples were taken from each of the two types of Astragalus.

According to the method of the present invention, the selected Astragalus samples are processed, and then separated by gel electrophoresis to obtain the electrophoresis figure of each Astragalus sample, as shown in Figure 1, in the figure, S is the sugar standard containing 6 polymerization degrees 1-12 are samples of Astragalus membranaceus, 13-24 are samples of Astragalus membranaceus, DP represents the degree of polymerization of monosaccharides, DP1-DP6 are monosaccharides, disaccharides, trisaccharides, disaccharides, pentasaccharides and hexasaccharides, respectively . Import the electropherogram in Figure 1 into Quantity One software, after processing, get the fingerprint spectrum corresponding to the electropherogram, as shown in Figure 2, in the figure, Nos. 1-12 are samples of Astragalus mongolica, and Nos. 13-24 are membranes Astragalus viburnum sample. Import the fingerprint data in Figure 2 into SPSS16.0 for T-test analysis, and get Figure 3. From Figure 3, it can be seen that disaccharides have significant differences, that is, disaccharides are the main difference fragments for distinguishing Astragalus species, and * in the figure Glycan fragments representing significant differences.

Import the fingerprint data in Figure 2 into SMICA-P13.0 for analysis. The analysis results are shown in Figure 4, Figure 5 and Figure 6. 4, it can be seen that Astragalus mongolica and Astragalus membranaceus can be clearly separated; Fig. 5 is the verification diagram of the PLS-DA analysis model, in which R ² is the value of the explanation model, and Q ² is the value of the prediction model. From Fig. 5, it can be seen that PLS-DA The DA model was verified and established, and this model can be used to search for differential sugar fragments in the identification of Astragalus species; Figure 6 is the loading diagram of differential sugar fragments. It can be seen from Figure 6 that the scattered points are far from the center, and the differences between different species of Astragalus membranaceus larger. The number of differential sugar fragments was counted, and Figure 7 was obtained. From Figure 7, it can be seen that trisaccharides and disaccharides are the main differential fragments for distinguishing Astragalus species.

Bring the optical density values in the fingerprint spectrum in Figure 2 into the standard curves of the disaccharide standard and trisaccharide standard respectively, and calculate the disaccharide and trisaccharide content in the Astragalus sample. Figure 8 and Figure 9 are the disaccharide standard and trisaccharide respectively. The standard curve figure of the three sugar standard substance, in the figure, y is the optical density value, x is the concentration value, and R ² is the correlation coefficient, and the calculated results are shown in Table 1;

Astragalus sample number Disaccharide content (mg/mL) Trisaccharide content (mg/mL) Disaccharide content/Trisaccharide content 1 0.873 3.310 0.264 2 0.809 3.088 0.262 3 0.985 3.384 0.291 4 0.945 3.270 0.289 5 0.681 3.013 0.226 6 0.783 3.058 0.256 7 0.864 2.958 0.292 8 0.748 2.662 0.281 9 0.857 2.866 0.299 10 0.685 2.991 0.229 11 0.795 2.978 0.267 12 0.774 2.660 0.291 13 1.026 3.167 0.324 14 1.102 3.158 0.349 15 1.298 3.197 0.406 16 1.096 3.036 0.361 17 1.112 3.038 0.366 18 1.372 3.330 0.412 19 1.016 2.509 0.405 20 1.134 2.961 0.383 twenty one 1.028 2.763 0.372 twenty two 1.123 2.894 0.388 twenty three 1.231 2.959 0.416 twenty four 1.035 2.974 0.348

Table 1

Table 1 shows the contents and ratios of disaccharides and trisaccharides of 24 Astragalus samples; as can be seen from Table 1, the contents of disaccharides in No. 1-12 samples of Astragalus mongolica are less than 1.0 mg/mL, and the content of disaccharides and trisaccharides The ratios of the contents were all less than 0.3; the disaccharide contents in samples of Astragalus membranaceus No. 13-24 were all greater than 1.0 mg/mL, and the ratios of the disaccharide content to the trisaccharide content were all greater than 0.3.

Therefore, the present invention can use the content of disaccharides and trisaccharides as the standard for distinguishing Astragalus species, that is, when the disaccharide content is less than 1.0 mg/mL, and when the disaccharide content/trisaccharide content is less than 0.3, it is Astragalus mongolica; When the sugar content > 1.0 mg/mL, and the disaccharide content/trisaccharide content > 0.3, it is Astragalus membranaceus.

Claims (5)

1. a kind of method for differentiating astragalus mongolicus and Astragalus membranacus, it is characterised in that comprise the following steps：

1) pretreatment of raw material：The dry Radix Astragali is ground into after powder 100 mesh sieves excessively and obtains Radix Astragali fine powder, it is standby；

2) Thick many candies are extracted：Take above-mentioned Radix Astragali fine powder add water after at a temperature of 100 DEG C refluxing extraction 1h, be down to after room temperature centrifuge Take supernatant；Residue adds water repetition and extracted 1 time, takes supernatant, merges supernatant；Concentration, by the supernatant of merging with 95% It is that 80%, 3000r/min centrifuges 10min that ethanol, which is adjusted to alcohol content, merges precipitation, is freeze-dried to obtain polysaccharide coarse powder；

3) purified polysaccharide：By step 2) obtained polysaccharide coarse powder is soluble in water, obtains polysaccharide meal solution, the polysaccharide coarse powder with The mass ratio of water is 1:200, into polysaccharide meal solution add concentration be 10.6% potassium ferrocyanide solution and concentration be 21.9% acetic acid zinc solution, the acetic acid zinc solution that the potassium ferrocyanide solution and concentration that the concentration is 10.6% are 21.9% Addition be the 10% of polysaccharide meal solution volume, stand 30min after shaking, centrifugation obtains purified polysaccharide, freeze, it is standby With；

4) polysaccharide sour water solution：Take step 3) obtained purified polysaccharide 10mg, the μ L of 0.5mol/L trifluoroacetic acids 500 are added, are mixed, with Mixed liquor is placed in 90 DEG C of insulation 1h afterwards, after reaction terminates, drying instrument is blown with nitrogen and dries polysaccharide acid hydrolysis products, then add suitable Methanol cleaning hydrolysate is measured, continues nitrogen drying；

5) derivatization of polysaccharide acid hydrolysis products, three saccharides and two saccharides：Take three saccharide 6mg, two Standard for Sugars Product 2mg and step 4) in obtained polysaccharide acid hydrolysis products, be separately added into 200 μ L NaCNBH3 solution, then be separately added into 200 μ L ANTS derivatization reagents, are vortexed, and mix；After 37 DEG C of isothermal reaction 15h, three kinds of products are dried up with nitrogen, are dissolved in 1mL6mol/L urea liquid, is made the derivatization sample of three saccharides, two saccharides and polysaccharide acid hydrolysis products, its In the derivatization samples of three saccharides and two saccharides be diluted to no less than 6 concentration respectively with 6mol/L urea liquid The derivatization sample of gradient；

6) derivatization sample electrophoresis are analyzed：Take step 5) in polysaccharide acid hydrolysis products derivatization sample, three saccharide derivatizations Sample and two saccharide derivatization samples, separate each derivatization sample, separation gel is concentration using vertical slab gel electrophoresis For 30% polyacrylamide gel, concentration glue is the polyacrylamide gel that concentration is 8%, and electrophoretic buffer is pH8.0- 9.0th, concentration 0.1-0.2mol/L Tris-boric, applied sample amount is 1-3 μ L, and gel is imaged under the conditions of UV300nm, is obtained many The electrophoretogram of saccharic acid hydrolysate derivatization sample, three saccharide derivatization samples and two saccharide derivatization samples；

7) standard curve of three saccharides and two saccharides is drawn：By polysaccharide acid hydrolysis products derivatization sample, trisaccharide mark The electrophoretogram of quasi- product derivatization sample and two saccharide derivatization samples imports Quantity One softwares, after treatment, The OD value of polysaccharide acid hydrolysis products and three saccharides and two saccharides is obtained, with three saccharides and two saccharides OD value be that ordinate, concentration are abscissa, draw standard curve；

8) discriminating of astragalus mongolicus and Astragalus membranacus：Bring the OD value of polysaccharide acid hydrolysis products into step 7) in standard it is bent Line, calculates the content of trisaccharide and disaccharides in astragalus polyose, and differentiate astragalus mongolicus and Astragalus membranacus according to following standard：

As two sugared content ＜ 1.0mg/mL, and it is astragalus mongolicus during two sugared contents/tri- sugared content ＜ 0.3；

As two sugared content ＞ 1.0mg/mL, and it is Astragalus membranacus during two sugared contents/tri- sugared content ＞ 0.3.

2. a kind of method for differentiating astragalus mongolicus and Astragalus membranacus according to claim 1, it is characterised in that：Step 2) in The mass ratio of the Radix Astragali fine powder and water is 1:100.

3. a kind of method for differentiating astragalus mongolicus and Astragalus membranacus according to claim 1, it is characterised in that：Step 2) in The mass ratio of the residue and water is 1:80.

4. a kind of method for differentiating astragalus mongolicus and Astragalus membranacus according to claim 1, it is characterised in that：Step 6) in The solvent of preparing of separation gel and concentration glue is pH8.0~9.0,0.1~0.2mol/L of concentration Tris-boric buffer solutions.

5. a kind of method for differentiating astragalus mongolicus and Astragalus membranacus according to claim 1, it is characterised in that：Step 6) in Electrophoretic separation process be carried out in two steps, first using 60-80V separation voltage electrophoretic separation 40-60min, then use 100-200V separation voltage electrophoretic separation 100-120min.