CN106083979A - Cycloastragenol extract and preparation method and application thereof - Google Patents

Abstract

The invention provides a cycloastragaloside alcohol extract, which is prepared by the following method: extracting astragalus coarse powder with an alkaline alcohol aqueous solution with a pH value of 11-13 to obtain a crude astragaloside extract; enriching and purifying the obtained crude astragaloside extract with a macroporous resin to obtain an astragaloside extract; Smith degradation of the obtained astragaloside extract to obtain the cycloastragaloside alcohol extract; the preparation of the cycloastragaloside alcohol extract of the invention obtains the most suitable extraction method through multiple tests on the pH value, sodium periodate dosage and methanol concentration in the extraction conditions, and the method has good reproducibility and is simple and easy to operate; the _{cycloastragaloside} alcohol extract prepared by the invention can significantly slow down the aging damage caused by _H2O2 to PC12 cells and improve cell viability, indicating that the cycloastragaloside alcohol extract obtained by the preparation method of the invention has certain anti-aging activity and can be used in the preparation of anti-aging drugs and health products.

Description

A cycloastragenol extract and its preparation method and application

(1) Technical field

The invention relates to a cycloastragenol extract, a preparation method and application of the extract, and belongs to the field of medicine.

(2) Background technology

At present, the aging population of countries around the world is accelerating. According to statistics, in 2012, the elderly population aged 65 and above in my country accounted for 9.4% of the total population. Double the number of elderly people in 2012. In such a severe situation, human beings are facing enormous challenges. Aging and anti-aging have become a hot topic in medical discussion today. With the improvement of people's living standards and the continuous strengthening of the concept of returning to green life, how to effectively prevent aging and improve the quality of life has become an urgent problem that people need to solve. Therefore, people's demand for anti-aging related green health care products and plant extracts continues to increase.

In recent years, the discovery of telomerase (Telomerase) and the proposal of "Telomere hypothesis" have greatly promoted the research on aging. Telomere is a specific structure composed of DNA and protein at the end of chromosomes, which plays a role in protecting the integrity of chromosomes. Studies have shown that telomeres gradually shorten with the increase in the number of cell divisions, and eventually lose their ability to protect chromosomes, resulting in cell aging . At the same time, telomerase is a reverse transcription DNA polymerase that maintains the length of telomeres, so it plays an important role in cell proliferation and aging. By activating telomerase activity, delaying the shortening of telomeres can play a significant role. Anti-aging effects, therefore, effective telomerase activators have also become the focus of current research.

As a traditional Chinese medicine, studies have shown that Astragalus has anti-oxidation, anti-aging, anti-tumor, anti-viral and other effects. Astragaloside is one of the effective components of Astragalus, among which Astragaloside IV is often used as the main index of quality control of Astragalus medicinal materials. It has been reported that cycloastragenol (Cycloastragenol, CAG) is the aglycone of most astragalosides, and has a significant telomerase activation effect, so it has a series of medicinal values such as anti-aging, anti-virus, and anti-depression. It has been reported that astragaloside IV is mostly prepared by hydrolysis. The preparation methods include acid hydrolysis, Smith degradation, enzymatic hydrolysis, etc., wherein Smith degradation is relatively short because of its mild reaction conditions and easy control. At the same time, periodate oxidation reaction is a kind of Selective reactions, thus having a certain specificity, are often used as a method for the preparation of CAG. Zhou Xianli et al. invented a preparation method and application of CAG. Using astragaloside IV as raw material, the Smith degradation method was used to crudely extract CAG, and then silica gel column chromatography, recrystallization and other methods were used to obtain CAG with a purity of more than 95%. . Feng et al. optimized the degradation conditions of Smith, including the dosage of sodium periodate, the concentration of methanol, and the dosage of sodium borohydride, etc., to obtain a high-purity CAG extract.

So far, most of the work has focused on how to obtain high-purity CAG and what biological activity CAG single products have, and they are all based on astragaloside IV monomers, and there are very few related studies on cycloastragenol extracts. Preliminary studies have shown that the preparation of CAG extract from Radix Astragalus contains a large number of by-products, some of which are structural analogs of CAG. Therefore, the present invention mainly conducts relevant research on CAG extract and its structural analogs, which will help promote CAG extract to the international market, and provide certain technical support and theoretical basis for the development of anti-aging related health care products and medicines.

(3) Contents of the invention

The object of the present invention is to provide a cycloastragenol extract and its preparation method and use. The preparation method of the present invention can effectively hydrolyze astragaloside IV in astragalus into cycloastragenol. The method has certain selectivity and can improve cycloastragenol. The content of astragalol is low, and the reaction conditions are easy to control and optimize, and the process is simple and easy to operate. Compared with the other two preparation methods reported at present - acid hydrolysis and enzymatic hydrolysis, the duration is shorter. The cycloastragenol extract prepared by the invention has anti-aging effect and can be used for preparing anti-aging medicine and health products.

To achieve the above object, the present invention adopts the following technical solutions:

A cycloastragenol extract prepared by the following method:

(a) Extract the coarse powder of Astragalus membranaceus (with a particle size of 20 mesh) with an aqueous alkaline alcohol solution with a pH value of 11-13, evaporate the solvent and dry to obtain the crude extract of astragaloside IV;

In step (a), specifically, the operation method of the extraction is: mix the coarse powder of Astragalus membranaceus with an aqueous alkaline alcohol solution with a pH value of 11-13 at a material-to-liquid mass ratio of 1:6-10, soak for 12-16 hours, and then Raise the temperature to reflux for extraction for 2-3 hours, filter to obtain the primary filtrate and primary filter residue; mix the obtained primary filter residue with an alkaline alcohol aqueous solution with a pH value of 11-13 at a material-to-liquid mass ratio of 1:8-10, and heat up to reflux for extraction for 2-3 hours. 3h, filter to obtain the secondary filtrate and secondary filter residue; discard the secondary filter residue, combine the primary filtrate and the secondary filtrate, evaporate the solvent under reduced pressure (45-60 ° C), and then freeze-dry (using a vacuum freeze dryer) , to obtain the crude extract of astragaloside IV;

Preferably described alkaline alcohol aqueous solution is the 75% (v/v) ethanol aqueous solution of pH value 11～13; More preferably described alkaline alcohol aqueous solution is the 75% (v/v) ethanol aqueous solution of pH value 13; Said alkaline alcohol aqueous solution is regulated and realized with sodium hydroxide;

(b) The astragaloside IV crude extract obtained in step (a) is enriched and purified by macroporous resin to obtain the astragaloside IV extract;

In step (b), specifically, the operation method for the enrichment and purification of the macroporous resin is: dissolving the crude extract of astragaloside in 2 to 10 times the mass of water to obtain the crude extract of astragaloside, which is added to In the chromatographic column filled with macroporous resin, after static adsorption for 4 to 6 hours, water with 5 to 20 times the volume of the resin column, 30% to 40% (v/v, preferably 30%) ethanol aqueous solution, 70% (v /v) Elution with ethanol water solution, collect 70% (v/v) ethanol water eluate, evaporate the solvent under reduced pressure (45-60° C.), and obtain astragaloside IV extract;

The macroporous resin is selected from one of ADS-17, AB-8, D101, HPD300, preferably D101 resin;

(c) performing Smith degradation on the astragaloside IV extract obtained in step (b) to obtain the cycloastragenol extract;

In step (c), specifically, the Smith degradation operation method is: add astragaloside IV extract and sodium periodate to pure water or 20% to 90% (v/v, preferably 20%) methanol aqueous solution In A, the oxidation reaction was carried out at 0-35°C for 11-13 hours, and then the reaction was quenched with ethylene glycol. The reaction liquid was first concentrated under reduced pressure, and then extracted with ethyl acetate. The solvent was evaporated from the extract, and the residue was dissolved in 40 %～90% (v/v, preferably 60%) methanol aqueous solution B, add sodium borohydride, carry out reduction reaction at 0～35°C for 3～5h, then adjust the pH of the reaction system to 1～5h with concentrated sulfuric acid (98wt%) 3. Hydrolyze at room temperature (20-30° C.) for 22-26 hours. After hydrolysis, the reaction solution is extracted with ethyl acetate, and the solvent is evaporated from the extract to obtain the cycloastragenol extract;

The mass ratio of the astragaloside IV extract to sodium periodate is 1:0.4-6, preferably 1:1;

The mass ratio of the residual substance to sodium borohydride is 1:1-5, preferably 1:1;

The recommended volume dosage of the pure water or methanol aqueous solution A is 120-200 mL/g based on the mass of astragaloside IV extract; the recommended volume dosage of the methanol aqueous solution B is 150-800 mL/g based on the mass of residual substances;

The methanol aqueous solution A and the methanol aqueous solution B have no special meanings, and the labels "A" and "B" are only used to distinguish the methanol aqueous solution used in different operation steps.

The content of cycloastragenol in the cycloastragenol extract prepared by the invention is 16.45%-32.58%.

The cycloastragenol extract prepared by the invention can be applied to the preparation of anti-aging medicines and health care products.

The beneficial effects of the present invention are:

(1) The preparation of cycloastragenol extract of the present invention obtains the most suitable extraction method by repeated tests to the pH value in the extraction conditions, the amount of sodium periodate, and the concentration of methanol, and the method has good reproducibility , easy to implement;

(2) The cycloastragenol extract prepared by the present invention can significantly slow down the aging damage caused by H ₂ O ₂ to PC12 cells and improve cell viability, suggesting that the cycloastragenol extract obtained by the preparation method of the present invention has certain anti-aging activity.

(4) Description of drawings

Fig. 1 is the liquid chromatogram of astragaloside IV extract in embodiment 17;

Fig. 2 is the liquid phase chromatogram of the extract of astragalus alcohol in embodiment 18;

Fig. 3-A, 3-B are the mass spectrograms of cycloastragenol extract based on HPLC-LTQ-Orbitrap-MS ⁿ technology in embodiment 19; Fig. 3-A represents the mass spectrogram of cycloastragenol extract under positive ion mode, Fig. 3-B represents the cycloastragenol extract mass spectrogram in negative ion mode;

Figure 4-A and 4-B are the MTT method in Example 20 to detect the influence of the extract of Astragalus cycloastragalus on PC12 cells; Figure 4-A shows the toxicity of the extract of Astragalus cycloastragalus to PC12 cells, and Figure 4-B shows the toxicity of Astragalus cycloastragalus extract to PC12 cells Protective effect of alcohol extract on PC12 cells induced by H ₂ O ₂ ; Note: * in the figure means P<0.05; ** means P<0.01;

Figure 5 is the effect of the extract of astragalus alcohol in Example 21 on the activity of β-galactosidase in PC12 cells; wherein, A represents the final staining situation of the control group, B represents the final staining situation of the modeling group, and C represents the low-concentration drug The staining situation of the protection group, D represents the staining situation of the medium-concentration drug protection group, E represents the staining situation of the high-concentration drug protection group; (↑) represents the stained PC12 senescent cells, and the scale bar indicates 50 μM;

Fig. 6 is the effect of the extract of astragalus in Example 22 on the telomerase activity of cells induced by H ₂ O ₂ ; Note: * in the figure indicates P<0.05; ** indicates P<0.01.

(5) Specific implementation methods

The present invention will be further described below through specific examples, but the protection scope of the present invention is not limited thereto.

Embodiment 1 The preparation method of the astragaloside IV crude extract of the present invention:

The Radix Astragali used complies with the relevant provisions of the 2010 edition of the Chinese Pharmacopoeia. Take 30 g of Astragalus membranaceus coarse powder, add 10 times of 75% ethanol with pH of 13 and soak overnight, use the same solution to reflux extract twice, each time for 2 hours, filter, combine the filtrates, evaporate the solvent under reduced pressure, and then freeze-dry to obtain astragaloside IV The crude extract was 0.8084g, and the content of astragaloside IV was determined to be 0.7399% by HPLC detection.

Embodiment 2 The preparation method of astragaloside IV crude extract of the present invention:

The Radix Astragali used complies with the relevant provisions of the 2010 edition of the Chinese Pharmacopoeia. Take 30 g of Astragalus membranaceus coarse powder, add 10 times of 75% ethanol with pH of 12 and soak overnight, use the same solution to reflux extract twice, each time for 2 hours, filter, combine the filtrates, evaporate the solvent under reduced pressure and then freeze-dry to obtain astragaloside IV The crude extract was 0.8080g, and the content of astragaloside IV was determined to be 0.7381% by HPLC detection.

Embodiment 3 The preparation method of the astragaloside IV crude extract of the present invention:

The Radix Astragali used complies with the relevant provisions of the 2010 edition of the Chinese Pharmacopoeia. Take 30 g of Astragalus membranaceus coarse powder, add 10 times of 75% ethanol with pH 11 and soak overnight, use the same solution to reflux extract twice, each time for 2 hours, filter, combine the filtrates, evaporate the solvent under reduced pressure and then freeze-dry to obtain astragaloside IV The crude extract was 0.9088g, and the content of astragaloside IV was determined to be 0.6596% by HPLC detection.

Embodiment 4 The preparation method of astragaloside IV extract of the present invention:

The astragaloside IV crude extract 3g prepared according to Example 1 was dissolved in 10 times the amount of water, passed through the resin column filled with D101 macroporous resin, and removed successively with 10 times the volume of the resin column volume and 30% aqueous ethanol solution. Large polar impurities, then eluted with 70% ethanol water solution, collected 70% ethanol water eluate, recovered ethanol, concentrated under reduced pressure to dry powder, obtained astragaloside IV extract, and determined the content of astragaloside IV through high performance liquid phase detection was 14.63%.

Embodiment 5 The preparation method of astragaloside IV extract of the present invention:

The astragaloside IV crude extract 3g prepared according to Example 1 was dissolved in 10 times the amount of water, passed through the resin column filled with D101 macroporous resin, and removed successively with 10 times the volume of the resin column volume and 35% aqueous ethanol solution. Large polar impurities, then eluted with 70% ethanol water solution, collected 70% ethanol water eluate, recovered ethanol, concentrated under reduced pressure to dry powder, obtained astragaloside IV extract, and determined the content of astragaloside IV through high performance liquid phase detection was 9.08%.

Embodiment 6 The preparation method of astragaloside IV extract of the present invention:

The astragaloside IV crude extract 3g prepared according to Example 1 was dissolved in 10 times the amount of water, passed through the resin column filled with D101 macroporous resin, and removed successively with 10 times the volume of the resin column volume of water and 40% ethanol aqueous solution Large polar impurities, then eluted with 70% ethanol water solution, collected 70% ethanol water eluate, recovered ethanol, concentrated under reduced pressure to dry powder, obtained astragaloside IV extract, and determined the content of astragaloside IV through high performance liquid phase detection was 9.91%.

Embodiment 7 The preparation method of cycloastragenol extract of the present invention:

Take 200 mg of the astragaloside IV extract obtained according to the method of Example 4, add 0.6 times the amount of sodium periodate, and carry out oxidation reaction at 25 ° C for 12 hours in a 60% methanol aqueous solution (30 mL) environment, add dropwise Alcohol (0.0781g) stopped the reaction; the reaction solution was concentrated under reduced pressure to remove methanol, extracted three times with an equal volume of ethyl acetate, the organic phases were combined, the solvent was removed, the residue was dissolved in 60% methanol aqueous solution (20mL), and Amount of sodium borohydride, reduction reaction at 25 ° C for 4 hours; adjust the pH of the reaction solution to 2 with concentrated sulfuric acid, and hydrolyze it at room temperature for 24 hours; The alcohol extract is 0.0263g, and the content of cycloastragenol as measured by HPLC is 20.11%.

Embodiment 8 The preparation method of cycloastragenol extract of the present invention:

Take 200 mg of the astragaloside IV extract obtained according to the method of Example 4, add 1 times the amount of sodium periodate, and carry out oxidation reaction at 25 ° C for 12 hours in a 60% methanol aqueous solution (30 mL) environment, add dropwise Alcohol (0.0985g) stopped the reaction; the reaction solution was concentrated under reduced pressure to remove methanol, extracted three times with an equal volume of ethyl acetate, the organic phases were combined, the solvent was removed, the residue was dissolved in 60% methanol aqueous solution (20mL), and Amount of sodium borohydride, reduction reaction at 25 ° C for 4 hours; adjust the pH of the reaction solution to 2 with concentrated sulfuric acid, and hydrolyze it at room temperature for 24 hours; The alcohol extract is 0.0171g, and the content of cycloastragenol as measured by HPLC is 25.44%.

Embodiment 9 The preparation method of cycloastragenol extract of the present invention:

Take 1000 mg of the astragaloside IV extract obtained according to the method of Example 4, add 1 times the amount of sodium periodate, and carry out oxidation reaction at 15° C. for 12 hours in a 90% methanol aqueous solution (112 mL) environment, and dropwise add ethylene glycol Alcohol (0.42g) stopped the reaction; the reaction solution was concentrated under reduced pressure to remove methanol, extracted three times with an equal volume of ethyl acetate, the organic phases were combined, the solvent was removed, the residue was dissolved in 60% aqueous methanol (100mL), and 3 times the weight of the residue was added The sodium borohydride was reduced at 15°C for 4 hours; the pH of the reaction solution was adjusted to 2 with concentrated sulfuric acid, and hydrolyzed at room temperature for 24 hours; the reaction solution was extracted with ethyl acetate, the organic phases were combined, and the organic solvent was removed to obtain cycloastragenol The extract is 0.062g, and the content of cycloastragenol as measured by HPLC is 25.05%.

Embodiment 10 The preparation method of cycloastragenol extract of the present invention:

Take 200 mg of the astragaloside IV extract obtained according to the method of Example 4, add 1 times the amount of sodium periodate, and carry out oxidation reaction at 35° C. for 12 hours in a 20% methanol aqueous solution (30 mL) environment, add dropwise Alcohol (0.1025g) stopped the reaction; the reaction solution was concentrated under reduced pressure to remove methanol, extracted three times with an equal volume of ethyl acetate, the organic phases were combined, the solvent was removed, the residue was dissolved in 60% aqueous methanol (20mL), and Amount of sodium borohydride was reduced at 35°C for 4 hours; pH of the reaction solution was adjusted to 2 with concentrated sulfuric acid, and hydrolyzed at room temperature for 24 hours; The alcohol extract is 0.0291g, and the content of cycloastragenol as measured by HPLC is 32.58%.

Embodiment 11 The preparation method of cycloastragenol extract of the present invention:

Take 200 mg of astragaloside IV extract obtained according to the method of Example 4, add 3 times the amount of sodium periodate, and carry out oxidation reaction at 10 ° C for 12 hours in a 60% methanol aqueous solution (30 mL) environment, add dropwise Alcohol (0.2772g) stopped the reaction; the reaction solution was concentrated under reduced pressure to remove methanol, extracted three times with an equal volume of ethyl acetate, the organic phases were combined, the solvent was removed, the residue was dissolved in 60% aqueous methanol (20mL), and Amount of sodium borohydride, reduction reaction at 5°C for 4 hours; adjust the pH of the reaction solution to 2 with concentrated sulfuric acid, and hydrolyze it at room temperature for 24 hours; The alcohol extract is 0.0254g, and the content of cycloastragenol as measured by HPLC is 23.07%.

Embodiment 12 The preparation method of cycloastragenol extract of the present invention:

Take 200 mg of the astragaloside IV extract obtained according to the method of Example 4, add 5 times the amount of sodium periodate, and carry out oxidation reaction at 15° C. for 12 hours in a 60% methanol aqueous solution (30 mL) environment, add dropwise Alcohol (0.42g) stopped the reaction; the reaction solution was concentrated under reduced pressure to remove methanol, extracted three times with an equal volume of ethyl acetate, the organic phases were combined, the solvent was removed, the residue was dissolved in 60% aqueous methanol (20mL), and Amount of sodium borohydride, reduction reaction at 10°C for 4 hours; adjust the pH of the reaction solution to 2 with concentrated sulfuric acid, and hydrolyze it at room temperature for 24 hours; extract the reaction solution with ethyl acetate, combine the organic phases, remove the organic solvent, and obtain Cycloastragalus The alcohol extract is 0.0329g, and the content of cycloastragenol as measured by HPLC is 20.71%.

Embodiment 13 The preparation method of cycloastragenol extract of the present invention:

Get 200 mg of astragaloside IV extract obtained according to the method of Example 4, add 5 times the amount of sodium periodate, in a pure aqueous solution (30 mL) environment, carry out oxidation reaction at 10 ° C for 12 hours, drop ethylene glycol (0.4364 g) Stop the reaction; the reaction solution is concentrated under reduced pressure to remove methanol, extracted three times with an equal volume of ethyl acetate, the organic phases are combined, the solvent is removed, the residue is dissolved in 60% aqueous methanol (20mL), and the residue is hydroborated with an amount equal to the weight of the residue Sodium, carry out reduction reaction at 10 ℃ for 4 hours; adjust the pH of the reaction solution to 2 with concentrated sulfuric acid, and hydrolyze it at room temperature for 24 hours; g, the cycloastragenol content measured by HPLC is 21.71%.

Embodiment 14 The preparation method of cycloastragenol extract of the present invention:

Take 200 mg of the astragaloside IV extract obtained according to the method of Example 4, add 5 times the amount of sodium periodate, and carry out oxidation reaction at 10° C. for 12 hours in a 20% methanol aqueous solution (30 mL) environment, add dropwise Alcohol (0.4360g) stopped the reaction; the reaction solution was concentrated under reduced pressure to remove methanol, extracted three times with an equal volume of ethyl acetate, the organic phases were combined, the solvent was removed, the residue was dissolved in 60% aqueous methanol (20mL), and Amount of sodium borohydride, reduction reaction at 10°C for 4 hours; adjust the pH of the reaction solution to 2 with concentrated sulfuric acid, and hydrolyze it at room temperature for 24 hours; extract the reaction solution with ethyl acetate, combine the organic phases, remove the organic solvent, and obtain Cycloastragalus The alcohol extract is 0.0274g, and the content of cycloastragenol as measured by HPLC is 24.17%.

Embodiment 15 The preparation method of cycloastragenol extract of the present invention:

Take 200 mg of the astragaloside IV extract obtained according to the method of Example 4, add 5 times the amount of sodium periodate, and carry out oxidation reaction at 3° C. for 12 hours in a 60% methanol aqueous solution (30 mL) environment, add dropwise Alcohol (0.4377g) stopped the reaction; the reaction solution was concentrated under reduced pressure to remove methanol, extracted three times with an equal volume of ethyl acetate, the organic phases were combined, the solvent was removed, the residue was dissolved in 20% aqueous methanol (20mL), and Amount of sodium borohydride, reduction reaction at 5°C for 4 hours; adjust the pH of the reaction solution to 2 with concentrated sulfuric acid, and hydrolyze it at room temperature for 24 hours; The alcohol extract is 0.0284g, and the content of cycloastragenol as measured by HPLC is 16.45%.

Embodiment 16 The preparation method of cycloastragenol extract of the present invention:

Take 200 mg of the astragaloside IV extract obtained according to the method of Example 4, add 5 times the amount of sodium periodate, and carry out oxidation reaction at 8° C. for 12 hours in a 60% methanol aqueous solution (30 mL) environment, add dropwise Alcohol (0.4506g) stopped the reaction; the reaction solution was concentrated under reduced pressure to remove methanol, extracted three times with an equal volume of ethyl acetate, the organic phase was combined, the solvent was removed, the residue was dissolved in 60% aqueous methanol (20mL), and A certain amount of sodium borohydride was reduced at 8°C for 4 hours; the pH of the reaction solution was adjusted to 2 with concentrated sulfuric acid, and it was hydrolyzed at room temperature for 24 hours; The alcohol extract is 0.0331 g, and the content of cycloastragenol as measured by HPLC is 18.80%.

Determination of Astragaloside IV Content in Example 17 Astragaloside IV Extract:

1 Chromatographic conditions

Chromatographic column Phenomenex C18 (4.6mm*250mm, 5μm); mobile phase acetonitrile (A)-0.3% formic acid water (B); flow rate 0.5mL/min; column temperature 35℃; injection volume 5μL; drift temperature 40℃; nitrogen Flow rate 1.5L/min; elution gradient as follows: 0-15min, 66%B; 15-20min, 66%-55%B; 20-25min, 55%-50%B; 25-35min, 50%-49% B; 35-40min, 49%-30%B; 40-45min, 30%-0%B.

2 Preparation of the test solution

Take 0.8084 g of the astragaloside IV extract prepared according to Example 1 in a 50 mL volumetric flask, constant volume with methanol, and ultrasonically dissolve as the test solution.

3 Preparation of reference solution

Accurately weigh 1.01mg of astragaloside IV reference substance, put it in a 1mL volumetric flask, add methanol to dissolve it and dilute to the mark, and use it as the reference substance solution.

4 System adaptability test

The test sample was injected with 5 μL for measurement, and the results showed that the retention time of astragaloside IV was about 27 minutes, and the chromatographic peak of astragaloside IV in the sample could be completely separated from other impurity peaks, so it could be seen that the sample contained astragaloside IV. The liquid chromatogram of astragaloside IV extract is shown in Figure 1.

5 Preparation of standard curve and investigation of linear range

Take the reference solution and inject 3 μL, 5 μL, 7 μL, 10 μL, 15 μL in sequence, and measure the peak area value according to the above chromatographic conditions. For regression, draw a standard curve. The experimental data was subjected to linear regression, and the regression equation was obtained as Y=1.3019X+7.3249, R2=0.9984, and the results are shown in Table 1. Experiments show that: the natural logarithm of the injection volume of the astragaloside IV reference substance in the range of 3.03μg-15.15μg has a good linear relationship with the natural logarithm of the peak area.

Table 1 Astragaloside IV linear range investigation table

Determination of Astragaloside IV in 6 samples

Take the test sample prepared according to Examples 1-6, and prepare the test sample solution with appropriate concentration respectively according to the above-mentioned test method for detection, and the results are shown in Table 2.

Determination of astragaloside IV content in the sample of Table 2

Determination of cycloastragenol content in embodiment 18 cycloastragenol extract:

1 Chromatographic conditions

Chromatographic column Phenomenex C18 (4.6mm*250mm, 5μm); mobile phase acetonitrile (A)-0.3% formic acid water (B); flow rate 0.5mL/min; column temperature 35℃; injection volume 5μL; drift temperature 40℃; nitrogen Flow rate 1.5L/min; elution gradient as follows: 0-10min, 50%-40%B; 10-20min, 40%-30%B; 20-30min, 30%-25%B; 30-35min, 25%B -10% B; 35-40 min, 10%-0% B.

2 Preparation of the test solution

Take 15 mg of the CAG extract prepared according to Example 9 in a 5 mL volumetric flask, determine the solution with 70% methanol aqueous solution, and ultrasonically dissolve it as the test solution.

3 Preparation of reference solution

Accurately weigh 2.04mg of cycloastragenol reference substance, put it in a 2mL volumetric flask, add 70% methanol aqueous solution to dissolve it and dilute to the mark, shake well, and use it as cycloastragenol reference substance stock solution. Precisely draw 200 μL of the stock solution into a 1mL volumetric flask, add 70% methanol aqueous solution to dilute to the mark, dissolve it by ultrasonic, and use it as the reference substance solution.

4 System adaptability test

The test sample was injected with 5 μL for measurement, and the results showed that the retention time of cycloastragenol was about 19 minutes, and the chromatographic peak of cycloastragenol in the sample could be completely separated from other impurity peaks, so it could be seen that the sample contained cycloastragenol. The liquid chromatogram of cycloastragenol extract is shown in Figure 2.

5 Preparation of standard curve and investigation of linear range

Accurately draw 10 μL, 50 μL, 100 μL, 200 μL, and 500 μL of the reference substance solution respectively, place them in a 1mL volumetric flask, add 70% methanol water to dilute to the mark, shake well, and measure the peak area according to the above chromatographic conditions. The logarithm is the abscissa, and the natural logarithm of the peak area is the ordinate to perform regression and draw the standard curve. The experimental data was subjected to linear regression, and the regression equation was Y=1.237X+7.2748, R ² =0.9986. The results are shown in Table 3. Experiments show that: the natural logarithm of the cycloastragenol reference substance in the injection volume of 0.255 μg-5.100 μg has a good linear relationship with the natural logarithm of the peak area.

Table 3 cycloastragenol linear range investigation table

Determination of the content of cycloastragenol in 6 samples

Take the test samples prepared according to Examples 7-16, and prepare test sample solutions with appropriate concentrations according to the above-mentioned test method for testing. The results are shown in Table 4.

Determination of cycloastragenol content in the sample of table 4

The composition speculation of embodiment 19 cycloastragenol extracts:

The invention adopts the HPLC-LTQ-Orbitrap-MSn technology to obtain the mass spectrogram of the cycloastragenol extract sample, and performs structural identification according to the spectrogram and the comparison of some standard products. Due to the limitation of the types of standard species, only a rough guess is made on the components of the extract for reference in further research.

1 Preparation of standard solution

Take an appropriate amount of astragaloside IV, cycloastragenol and formononetin standard substances in a 1.5mL centrifuge tube, dissolve them ultrasonically with 800μL of 70% methanol aqueous solution, and set aside.

2 Preparation of sample solution

Considering the diversity of compound types in the extract, the cycloastragenol extract prepared in Example 9 containing more components was selected as a sample, and about 10 mg was weighed, and the volume was adjusted to 1 mL with 70% aqueous methanol as the sample solution .

3 Mass Spectrometry Conditions

HPLC-LTQ-Orbitrap-MS ⁿ mass spectrometry conditions: Electrospray ion source (ESI), positive ion mode, sheath gas pressure 303.4kPa, auxiliary gas pressure 34.5kPa, spray voltage 2.5kV, capillary temperature 350°C, tube lens voltage 96V , the capillary voltage is 35V; the sample is firstly scanned by FT, the mass scanning range is m/z 120-1200, the resolution is FS 30000, MS27500, the secondary mass spectrometer adopts dynamic data dependent scan (DDS), and the collision energy setting If it is 25%-35%, the highest abundance peak of the upper level is taken for collision-induced dissociation (CID) fragment scanning and detected by ion trap (dynode). Data analysis and processing were performed by Xcalibur software.

4 Experimental results and analysis

The mass spectrograms of the extract in positive and negative ion modes are shown in Figures 3-A and 3-B.

After mass spectrometry data analysis, the mass spectrometry data of related compounds in a large number of literatures and the mass spectrometry data of existing standards were compared, and some peak components in the extract were initially identified. The identification results are shown in Table 5 and Table 6.

Table 5 Identification of peak components in mass spectrum in positive ion mode

Table 6 Identification of peak components in mass spectrogram in negative ion mode

Example 20 The effect of CAG extract on the activity of PC12 cells damaged by H ₂ O ₂ oxidation:

1 Experimental materials

1.1 Drugs: The cycloastragenol extract is prepared by the preparation method described in Example 9, with a purity of 25.05%. Before use, prepare a 200mg/mL stock solution with analytical grade DMSO, and sterilize it for later use.

1.2 Materials: PC12 cells were donated by Professor Li Ping of China Pharmaceutical University. DMEM medium, fetal bovine serum (FBS), horse serum (HS), trypsin, thiazolyl blue (MTT) powder, and PBS were purchased from Yujie Biotechnology Co., Ltd.; hydrogen peroxide and DMSO were purchased from Sigma.

1.3 Instruments and instruments: sterile room, ultra-clean table, low-speed centrifuge, ordinary optical microscope, alcohol lamp, petri dish, centrifuge tube, microplate reader, oscillator.

2 Experimental steps

2.1 Cell culture: Culture PC12 cells in DMEM medium containing 10% fetal bovine serum, 5% horse serum and 1% double antibody (penicillin-streptomycin mixture), placed in 5% CO ₂ , saturated humidity, 37 Incubate in an incubator at ℃. When the cells were about 80% attached to the wall, they were digested and passaged with 0.25% trypsin.

2.2 Cytotoxicity test of cycloastragenol extract

2.2.1 Pre-treatment of cell detection: Make PC12 cells with a density of 2.0*10^5 cells/mL when they adhere to the wall at about 80%, and inoculate them in a 96-well cell culture plate at a volume of 200 μL per well , cultured in the above incubator until about 80% of the cells adhered to the wall and processed in groups. Set up control group, zeroing group and administration group (administration concentration is: 0.01mg/mL, 0.1mg/mL, 0.5mg/mL, 0.8mg/mL, 1.0mg/mL, 2.0mg/mL, 5.0mg /mL, 10mg/mL). MTT assay was performed 24 hours after administration.

2.2.2 MTT detection: Add 20 μL MTT solution to each well, continue to cultivate for 4 hours, absorb the culture medium, add 150 μL DMSO to each well, shake for 15 minutes, measure the OD value with a detection wavelength of 570 nm and a reference wavelength of 630 nm with a microplate reader, according to The OD value was used to calculate the cell viability, and the calculation formula was as follows:

According to the cell viability, the cell inhibition rate=1-cell viability was calculated. The drug toxicity curve was drawn according to the cell inhibition rate.

2.3 Protective effect experiment of cycloastragenol extract

2.3.1 Pre-treatment of cells for detection: pre-administration treatment is the same as that described in 2.2. Set up the control group, modeling group, zeroing group and administration group (the administration concentration is 0.001mg/mL, 0.01mg/mL, 0.05mg/mL, 0.1mg/mL, 0.5mg/mL, 1mg/mL) . After 12 hours of administration, the model was established with H ₂ O ₂ solution with a concentration of 800 μM prepared in DMEM, and the MTT test was carried out 6 hours later.

2.3.2 MTT detection: the steps are the same as those described in 2.2. Finally, the histogram of the protective effect of the drug was drawn according to the cell viability.

3. Experimental results

The results showed that the IC50 value of the cycloastragenol extract prepared in Example 9 on PC12 cells was 1.39 mg/mL. In the experimental concentration range of 0.001 mg/mL-0.1 mg/mL, the extract showed a certain protective effect on PC12 cells induced by H ₂ O ₂ . The results are shown in Figures 4-A and 4-B.

Example 21 Effect of CAG extract on PC12 cell β-galactosidase activity:

1. Experimental materials

1.1 Drugs: as described in Example 20.

1.2 Materials: PC12 cells were donated by Professor Li Ping of China Pharmaceutical University. DMEM medium, fetal bovine serum (FBS), horse serum (HS), trypsin, and PBS were purchased from Yujie Biotechnology Co., Ltd.; hydrogen peroxide and DMSO were purchased from Sigma; aging-related β-galactosidase staining kit was purchased From Biyuntian Biotechnology Co., Ltd.

1.3 Instruments and instruments: sterile room, ultra-clean table, low-speed centrifuge, ordinary optical microscope, alcohol lamp, petri dish, centrifuge tube, oscillator, constant temperature water bath.

2 Experimental steps

2.1 Cell culture: same as described in Example 20.

2.2 Pre-treatment for cell detection: Make PC12 cells with a density of 4.0*10^5 cells/mL when they adhere to the wall at about 80%, and inoculate them in a 24-well cell culture plate at a volume of 800 μL per well. The cells were cultured in the above incubator until about 80% of the cells adhered to the wall. A control group, a modeling group and an administration group were set up (administration concentrations were 0.001 mg/mL, 0.05 mg/mL, and 0.1 mg/mL in turn). Twelve hours after administration, the model was established with H ₂ O ₂ solution with a concentration of 800 μM prepared in DMEM, and β-galactosidase staining test was carried out 6 hours later.

2.3 Detection of senescence-related β-galactosidase activity: a. Aspirate the cell culture medium, wash once with PBS, add 250 μL β-galactosidase staining fixative to each well, and fix at room temperature for 15 minutes; b. Aspirate the cell fixative, Wash three times with PBS, each time for three minutes; c. Aspirate off the PBS, add 250 μL of staining working solution to each well (staining solution A: staining solution B: staining solution C: X-Gal solution = 1:1:93:5; v /v/v/v); d.seal the 24-well plate with parafilm to prevent evaporation, and incubate overnight in a 37°C water bath; e. absorb the staining working solution, wash once with PBS, and observe under an ordinary optical microscope.

3 Experimental results

The results showed that compared with the cells in the control group, the staining of the cells treated with H ₂ O ₂ was more obvious. 1. The number of stained cells under high dosage concentration was lower than that of the model group, and the number of stained cells in the dosage group showed a downward trend with the increase of dosage concentration. Since senescent cells express β-galactosidase with high enzyme activity at pH 6.0, and the kit uses X-Gal as a substrate, it will produce dark blue under the catalysis of senescence-specific β-galactosidase The product, the stained cells, are cells in a senescent state. It can be seen that the extract of cycloastragenol exhibited certain anti-aging activity on PC12 cells induced by H ₂ O ₂ . The results are shown in Figure 5.

Example 22 The effect of cycloastragenol extract on the telomerase activity of PC12 cells:

1 Experimental materials

1.1 Drugs: as described in Example 20.

1.2 Materials: PC12 cells were donated by Professor Li Ping of China Pharmaceutical University. DMEM medium, fetal bovine serum (FBS), horse serum (HS), trypsin, and PBS were purchased from Yujie Biotechnology Co., Ltd.; hydrogen peroxide and DMSO were purchased from Sigma; RIPA lysate (strong), PMSF (100mM) were purchased From Biyuntian Biotechnology Co., Ltd.; rat telomerase (TE) enzyme-linked immunoassay (ELISA) kit was purchased from Shanghai Jianglai Biological Co., Ltd.

1.3 Instruments and instruments: sterile room, ultra-clean table, low-speed centrifuge, inverted microscope, alcohol lamp, petri dish, centrifuge tube, oscillator, low-temperature centrifuge, ice pack.

2 Experimental steps

2.1 Cell culture: same as described in Example 20.

2.2 Pre-treatment of cell detection: Make PC12 cells with a density of 5.0*10^5 cells/mL when they are adhered to the wall at about 80%, inoculate them in a 6-well cell culture plate at a volume of 2 mL per well, and The cells were cultured in the above incubator until about 80% of the cells adhered to the wall. A control group, a positive control group (concentration of 2 μM cycloastragenol monomer) and a cycloastragenol extract administration group (administration concentrations of 0.01 mg/mL, 0.1 mg/mL, and 0.5 mg/mL were set up). After 12 hours of administration, the model was established with H ₂ O ₂ solution with a concentration of 800 μM prepared in DMEM, and the cell lysate was prepared 6 hours later.

2.3 Preparation of cell lysate: a. Aspirate the cell culture medium and wash twice with PBS; b. Aspirate the PBS, add 400 μL of the prepared lysate to each well, shake for 30 minutes; c. Pipette repeatedly with a 1 mL pipette , pipette the lysate into a 1.5mL centrifuge tube, centrifuge at 4°C, 1500rpm, 15min; d. Pipette 300μL/tube of the supernatant into a 1.5mL centrifuge tube, seal, store in dry ice, and send for inspection.

3 Experimental results

The results show that the telomerase activity of the positive control group and the administration group is significantly higher than that of the control group, and the telomerase activity of the administration group increases with the increase of the administration concentration. It can be seen that within the test concentration range, the implementation The cycloastragenol extract prepared in Example 9 has a relatively obvious telomerase activation effect, suggesting that the extract has a good anti-aging effect. The results are shown in Figure 6.

Claims (9)

1. a cycloastragenol extract, is characterized in that, described cycloastragenol extract is prepared by following method: (a) extract the coarse powder of astragalus with an aqueous alkaline alcohol solution with a pH value of 11 to 13, evaporate the solvent and dry to obtain the crude extract of astragaloside IV; (b) dissolving the astragaloside IV crude extract obtained in step (a) in 2 to 10 times by mass water to obtain the astragaloside IV crude extract, which is added to a chromatographic column filled with a macroporous resin for static adsorption for 4 After ~6h, use 5 ~ 20 times the volume of the resin column in water, 30% ~ 40% ethanol water solution by volume fraction, and 70% ethanol water solution by volume fraction to elute sequentially, collect the 70% ethanol water eluate by volume fraction, and evaporate under reduced pressure. Solvent, obtain astragaloside IV extract; Described macroporous resin is selected from the one in ADS-17, AB-8, D101, HPD300; (c) Add the astragaloside IV extract and sodium periodate obtained in step (b) into pure water or methanol aqueous solution A with a volume fraction of 20% to 90%, and perform an oxidation reaction at 0 to 35°C for 11 to 13 hours, and then The reaction was quenched with ethylene glycol, the reaction solution was first concentrated under reduced pressure, and then extracted with ethyl acetate, the extract was evaporated to remove the solvent, the residue was dissolved in 40% to 90% methanol aqueous solution B, and sodium borohydride was added. Carry out the reduction reaction at 0-35°C for 3-5 hours, then use concentrated sulfuric acid to adjust the pH of the reaction system to 1-3, hydrolyze at room temperature for 22-26 hours, after hydrolysis, the reaction solution is extracted with ethyl acetate, and the extract is evaporated to remove the solvent to obtain the cycloastragenol extract; The mass ratio of the astragaloside IV extract to sodium periodate is 1:0.4-6; the mass ratio of the residue to sodium borohydride is 1:1-5. 2. cycloastragenol extract as claimed in claim 1, is characterized in that, in step (a), the operating method of described extraction is: the basic alcoholic aqueous solution of astragalus coarse powder and pH value 11～13 is fed Liquid mass ratio 1: 6~10 mixed, soaked for 12~16h, then raised to reflux for extraction for 2~3h, filtered to obtain primary filtrate and primary filter residue; the obtained primary filter residue and alkaline alcohol aqueous solution with pH value 11~13 were used as material The liquid mass ratio is 1: 8-10, and the temperature is raised to reflux for extraction for 2-3 hours, and then filtered to obtain the secondary filtrate and secondary filter residue; the secondary filter residue is discarded, the primary filtrate and the secondary filtrate are combined, and the solvent is evaporated under reduced pressure. Freeze-dry to obtain the crude extract of astragaloside IV. 3. the cycloastragenol extract as claimed in claim 1 or 2, is characterized in that, described alkaline alcohol aqueous solution is the volume fraction 75% ethanol aqueous solution of pH value 11～13, and described alkaline alcohol aqueous solution uses Sodium hydroxide is adjusted to achieve. 4. cycloastragenol extract as claimed in claim 1, is characterized in that, in step (b), described macroporous resin is D101 resin. 5. cycloastragenol extract as claimed in claim 1, is characterized in that, in step (c), the volume dosage of described pure water or methanol aqueous solution A is 120～200mL/ in terms of the quality of astragaloside IV extract. g. 6. The cycloastragenol extract according to claim 1, characterized in that, in step (c), the volumetric dosage of the aqueous methanol solution B is 150-800 mL/g based on the mass of the residual substance. 7. The cycloastragenol extract according to claim 1, characterized in that, in step (c), the mass ratio of the astragaloside IV extract to sodium periodate is 1:1. 8. Cycloastragenol extract as claimed in claim 1, characterized in that, in step (c), the mass ratio of the residual substance to sodium borohydride is 1:1. 9. the application of cycloastragenol extract as claimed in claim 1 in the preparation of anti-aging medicine and health products.