CN1199651C - Astragalus root valid part of Chinese herbal medicine for treating cardiovascular disease - Google Patents

Abstract

A kind of astragalus total saponins which has the effect of protecting myocardial injury, the ethanol extract of astragalus is dissolved in water, put on a macroporous resin column, eluted with water and ethanol containing water respectively, combined with 70%-98% ethanol eluate, evaporated to dryness to obtain Cream; dissolve the extract with methanol, add diatomaceous earth and mix well, the ratio of diatomaceous earth to extract is 3-1:1, extract with absolute ethanol under hot reflux, and evaporate the obtained extract to dryness to obtain saponin extract, which contains The total saponins reach 85%-90%, and the transfer rate of total saponins is 80%-85%.

Description

Effective parts of Astragalus membranaceus, a traditional Chinese medicine for preventing and treating cardiovascular diseases

technical field

The invention obtains high-purity total astragalus saponins with the effect of protecting myocardial damage through the improvement of the preparation method.

Background technique

Radix astragalus (Radix astragalus) is the dried root of Astragalus membranaceus (Fisch.) Bge.var mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge. of the leguminous plant Astragalus membranaceus (Fisch.) Bge. Spleen meridian, invigorating qi and solidifying the exterior, inducing diuresis to relieve toxins, evacuating pus, astringing sores and promoting granulation. Modern chemical research has proved that Astragalus contains saponins, polysaccharides, flavonoids, amino acids, phenolic compounds and trace elements. Astragaloside has a strong heart function and can be used to treat heart failure, and has a significant effect on the recovery of heart function after acute myocardial infarction and the scavenging of oxygen free radicals. Especially in recent years, astragalus has been used to treat viral myocarditis, and it has been found that astragalus can inhibit the replication of coxsackie virus and reverse the abnormal electrical activity of virus-infected myocardial cells. The protective effect of Astragalus proved that the main active ingredient of Astragalus is total saponins of Astragalus. However, the content of astragaloside is low, and the root contains a lot of sugar substances, so it is difficult to extract and separate.

Existing extraction and separation processes include: water extraction, alcohol extraction, resin adsorption, n-butanol extraction and supercritical CO ₂ method. In order to remove sucrose and monosaccharides in medicinal materials, cold soaking method is useful (see: sugar-free enrichment active ingredient extract and its products, CN1171950A); ).

Contents of the invention

The technical problem to be solved by the present invention is to obtain total saponins with high content and improved purity by improving the existing method for extracting and separating astragalosides from effective parts of astragalus, so as to ensure stable curative effect and have clearer quality control indicators.

In order to solve the above problems, the present invention provides the following technical solutions:

A kind of astragaloside is characterized in that available following method obtains:

1) Take the ethanol extract of Astragalus membranaceus, dissolve it in water, put it on a macroporous resin column, elute with water and ethanol containing water gradient, and combine the 70%-98% ethanol eluent to obtain the crude product of total saponins of Astragalus;

2) Alternatively, take the ethanol extract of Radix Astragali, dissolve it in water, first extract it with 0.1N NaOH-saturated n-butanol, concentrate the extract to an extract, then dissolve it in water, put it on a macroporous resin column, and mix it with water Ethanol gradient elution, combining 70% and 70%-98% ethanol eluents to obtain the crude product of astragalus total saponins;

3) Alternatively, take the ethanol extract of Astragalus membranaceus, dissolve it in methanol, add diatomaceous earth, mix well, extract with absolute ethanol under reflux, and evaporate the obtained extract to dryness to obtain crude total saponins of Astragalus membranaceus.

The astragaloside is characterized in that:

The crude product of total astragalosides obtained in 1) was dissolved in water, applied to a macroporous resin column again, gradient eluted with water and aqueous ethanol, and combined with 70%-98% ethanol eluents to obtain total astragalosides;

Alternatively, the crude product of total astragalosides obtained in 1) is dissolved in methanol, mixed with diatomaceous earth, extracted with absolute ethanol under reflux, and the obtained extract is evaporated to dryness to obtain total astragalosides;

Alternatively, the crude product of total astragalosides obtained in 2) is dissolved in methanol, mixed with diatomaceous earth, extracted with absolute ethanol under reflux, and the obtained extract is evaporated to dryness to obtain total astragalosides;

Alternatively, the crude product of total astragalosides obtained in 3) is dissolved in water, put on a macroporous resin column, gradient eluted with water and aqueous ethanol, and combined with 70%-98% ethanol eluents to obtain total astragalosides;

Alternatively, the crude product of astragalus total saponins obtained in 3) is dissolved in water, first extracted with n-butanol saturated with alkaline aqueous solution, such as extracted with 0.1N NaOH saturated n-butanol, concentrated extract to an extract, and then Dissolve in water, apply to a macroporous resin column, elute with water and ethanol gradient, combine 70%-98% ethanol eluent to obtain total saponins of astragalus.

The model of macroporous resin column used in the present invention is D101, AB-8; The weight ratio of ethanol extract extract and diatomaceous earth is 1: 1-3; The weight ratio of better ethanol extract extract and diatomite For 1:2.

A pharmaceutical composition for preventing and treating cardiovascular diseases, which contains a therapeutically effective amount of astragaloside according to any one of claims 1 or 2 and a pharmaceutically acceptable carrier.

The invention removes a large amount of carbohydrates in the astragalus through the selected macroporous resin column chromatography and certain elution conditions, and significantly increases the content of total saponins. If the macroporous resin column is applied twice, the total saponin content of the obtained extract can be significantly increased. Common impurities such as pigments in traditional Chinese medicines that affect the quality of preparations are generally removed by activated carbon, but the experimental data of the present invention shows that removing impurities such as pigments with activated carbon often greatly reduces the yield of astragaloside, and the present invention uses diatomite to mix Uniform, with absolute ethanol heat reflux adsorption method, the effect of removing impurities such as pigment is very obvious, and the yield of astragaloside is also high. The method of extracting total saponins in the process of the invention, the extraction solvent, the amount of solvent added, the reflux time and the number of times of extraction are obtained through orthogonal design and research, which ensures that the obtained astragalus total saponins has a high content and a transfer rate greater than 80%. Compared with the prior art, the invention has the advantages of high total saponin content, low cost, no organic solvent residue, no pollution to the environment in the production process and the like.

The astragaloside of the present invention has obvious effect of protecting myocardial injury. For pharmaceuticals, the purity of its therapeutic components is closely related to its efficacy. The present invention obtains astragaloside with a total saponin content greater than 80% by improving the existing method for extracting and separating astragaloside from the effective part of astragalus. The quality of the pharmaceutical composition is stable, and the quality control indicators are clear.

To study the optimal technology of astragalus total saponins (content greater than 80%), the preparation has the effect of protecting myocardial injury.

The research steps and detailed data are as follows:

1. Medicinal materials

The dry root of Astragalus membranaceus (Fisch.) Bge.varmongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge. is selected for use.

2. The influence of the extraction method of total saponins:

Take 50 g of astragalus medicinal material powder crushed into 40 meshes, accurately weighed, and extract according to the following methods respectively:

(1) Water extraction and alcohol precipitation: Take 50g of medicinal materials, add 200ml of distilled water, heat and extract 3 times, each time for 1.5h, centrifuge the water and liquid, combine the supernatant, concentrate to an appropriate amount, add 95% ethanol for precipitation, stand for 12h, and centrifuge , and the supernatants were combined and concentrated to obtain the extract.

(2) 50% alcohol extract: Take 50g of medicinal materials, add 200ml of 50% ethanol, heat and reflux at 90°C for 3 times, each time for 1.5h, combine the extracts, and concentrate to obtain the extract.

(3) 70% alcohol extract: Take 50g of medicinal materials, add 200ml of 70% ethanol, heat and reflux at 90°C for 3 times, each time for 1.5h, combine the extracts, and concentrate to obtain the extract.

(4) 95% ethanol extract: Take 50 g of medicinal materials, add 200 ml of 95% ethanol, heat and reflux at 90° C. for 3 times, each time for 1.5 h, combine the extracts, and concentrate to obtain an extract.

The experiment proves that the extract yield rate is 50% alcohol extract > 70% alcohol extract > 95% alcohol extract > water extraction and alcohol precipitation.

3. Separation, purification, concentration and drying process of total astragalus saponins

3.1 Separation and purification process

Take Astragalus mongolica or Astragalus membranaceus, crush the medicinal materials, heat and reflux extraction with 50-95% ethanol for 3 times, add 10 times the amount of 50-95% ethanol each time, and extract for 1.5h/time. The extracts were combined to obtain extract A.

Dissolve the extract A with an appropriate amount of distilled water, put it on a macroporous resin column, elute with distilled water, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 98% ethanol, and combine 70%-98% ethanol eluate was evaporated to dryness to obtain extract B.

Dissolve extract B in methanol, add diatomaceous earth and mix well, the ratio of diatomaceous earth to extract is 3-1:1, preferably 2:1, extract with absolute ethanol under reflux for 3 times, and the obtained extract is distilled Dry to obtain extract I. Calculated by astragaloside IV, the extract contains 85%-90% of total saponins, and the transfer rate of total saponins is 80%-85%.

If the extract I is dissolved in distilled water, put on a macroporous resin column, elute with distilled water, 20%, 30%, 40%, 50%, 60%, 70%, and 80% ethanol respectively, and combine 50%-80% Ethanol fractions were evaporated to dryness to obtain extract II. Calculated by astragaloside IV, the extract contained more than 98% of total saponins, and the transfer rate of total saponins was 75%-80%.

3.2 Concentration and drying process

Recover the solvent from the 70%-98% ethanol eluent under reduced pressure, and dry it in vacuum to obtain the total saponins.

Protective effect of total astragalus saponins of the present invention on cultured cardiomyocytes

1. Effect of Astragalus total saponins on calcium overload of cultured cardiomyocytes

(1) Effect of Astragalus total saponins on free calcium in cardiomyocytes

Total astragalosides intervening cells alone had no effect on intracellular free calcium ions ([Ca ²⁺ ] _i ), but when they acted together with isoproterenol, total astragalosides could inhibit [Ca ²⁺ ] _i induced by isoproterenol increase. Compared with isoproterenol, astragalus total saponins decreased [Ca ²⁺ ] _{i by} 39%, 40%, and 51% from low concentration to high concentration (10, 100, 200 mg·ml ^-1 ), respectively (P<0.05).

(2) Effects of total saponins of astragalus on the calcium loading of cardiomyocyte sarcoplasmic reticulum

Astragalus total saponins intervening cells alone has no effect on sarcoplasmic reticulum calcium load (Sarcoplasmic reticulum Calcium load), isoproterenol can increase sarcoplasmic reticulum calcium load by 145% (P<0.01), when it acts simultaneously with isoproterenol, Astragalus total saponins can inhibit the increase of sarcoplasmic reticulum calcium load caused by isoproterenol, from low concentration to high concentration respectively decreased by 11% (P>0.05), 35% (P<0.05), 51% (P<0.05) .

(3) Effect of total saponins of astragalus on Ca ²⁺ -ATPase activity of cardiomyocyte sarcoplasmic reticulum

Astragalus total saponins intervening cells alone can increase the activity of sarcoplasmic reticulum Ca ²⁺ -ATPase ^. increase, and it can be inhibited by 34% in the high concentration group (P<0.05).

2. Detection of Astragalus total saponins on the injury index (Troponin I) of cultured cardiomyocytes

Astragalus total saponins acted on the cultured cardiomyocytes, and the myocardial cell injury index in the supernatant - troponin I (cTnI) was detected, and there was no change in cTnI compared with the normal control group, indicating that astragalus total saponins had no damage to cardiomyocytes.

Detailed ways

Choose one of the following two kinds of Astragalus membranaceus (Fisch.) Bge. (Fisch.) Dried root of Bge. The traits and physical and chemical identification characteristics of the medicinal materials are in line with the content under the item "Astragalus" in the Pharmacopoeia of the People's Republic of China (1st Edition, 2000).

Example 1

Weigh 20kg of Radix Astragali, pulverize, pass through a 20-mesh sieve, add 8 times the amount of 50% ethanol, and extract at 80°C for 3 times, each time for .5 hours. The extracts were combined to obtain extract A. Dissolve the extract A with an appropriate amount of distilled water, extract three times with n-butanol saturated with 0.1N NaOH, combine the extracts to obtain extract B, dissolve the extract B with distilled water, put it on a macroporous resin column, and wash it with distilled water, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 98% ethanol elution, combined 70%-98% ethanol eluent, evaporated to dryness to obtain extract I, and According to the calculation of astragaloside IV, the extract contains 75.6% of total saponins, and the transfer rate of total saponins is 80.0%.

Example 2

Weigh 20 kg of Radix Astragali, pulverize, pass through a 20-mesh sieve, add 10 times the amount of 95% ethanol, and extract at 90° C. for 3 times, each time for 1.5 hours. The extracts were combined to obtain extract A. Dissolve the extract A with an appropriate amount of distilled water, extract three times with 0.1N NaOH-saturated n-butanol, combine the extracts to obtain extract B, dissolve the extract B with distilled water, put it on a macroporous resin column, and wash it with distilled water, 20 %, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 98% ethanol elution, combined 70%-98% ethanol eluent, evaporated to dryness to obtain extract C. Dissolve the extract C in methanol, add diatomaceous earth and mix well, the ratio of diatomaceous earth to extract is 2:1, extract 3 times with absolute ethanol under reflux, and evaporate the obtained extract to dryness to obtain extract I. According to the calculation of glycosides, the extract contains 85.2% of total saponins, and the transfer rate of total saponins is 72.5%.

Example 3

Weigh 20 kg of Radix Astragali, pulverize, pass through a 20-mesh sieve, add 10 times the amount of 70% ethanol, and extract at 80° C. for 3 times, each time for 1.5 hours. Combine the extracts to obtain the extract. Dissolve this extract with an appropriate amount of distilled water, put it on a macroporous resin column, elute with distilled water, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 98% ethanol respectively, and combine 70%-98% ethanol eluate was evaporated to dryness to obtain extract B. Dissolve extract B in methanol, add diatomaceous earth and mix well, the ratio of diatomaceous earth to extract is 2:1, extract 3 times with absolute ethanol under reflux, and evaporate the obtained extract to dryness to obtain extract I. According to the calculation of glycosides, the extract contains 83.4% of total saponins, and the transfer rate of total saponins is 81.2%.

Example 4

Weigh 20kg of Radix Astragali, pulverize, pass through a 20-mesh sieve, add 12 times the amount of 50% ethanol, and extract at 90°C for 3 times, each time for 1.5 hours. Combine the extracts to obtain the extract, dissolve the extract with an appropriate amount of distilled water, put it on a macroporous resin column, and add distilled water, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% , 98% ethanol elution, combined 40%-98% ethanol eluent, evaporated to dryness to obtain extract I, calculated astragaloside IV, this extract contains total saponins up to 74.8%, and the total saponin transfer rate is 85.1%.

Claims (5)

1, a kind of astragaloside, available following method obtains:

1) get the ethanol extraction extractum of the Radix Astragali, use water dissolution, last macroporous resin column, water and aquiferous ethanol gradient elution merge the 70%-98% ethanol elution, evaporate to dryness, the Radix Astragali total saponins crude product;

2) or, get the ethanol extraction extractum of the Radix Astragali, use water dissolution, earlier with the saturated n-butanol extraction of alkaline solution, concentrated extract is to the extractum shape, reuse water dissolution, last macroporous resin column, water and aquiferous ethanol gradient elution, merge the 70%-98% ethanol elution, evaporate to dryness gets the Radix Astragali total saponins crude product;

3) or, get the ethanol extraction extractum of the Radix Astragali, use dissolve with methanol, add kieselguhr and mix thoroughly, extract with the dehydrated alcohol hot reflux, gained extracting solution evaporate to dryness, the Radix Astragali total saponins crude product;

With the astragaloside crude product that obtains, handle through following method:

With 1) in obtain the Radix Astragali total saponins crude product, use water dissolution, go up macroporous resin column once more, water and aquiferous ethanol gradient elution merge the 70%-98% ethanol elution, evaporate to dryness, Radix Astragali total saponins;

Perhaps, with 1) in obtain the Radix Astragali total saponins crude product, use dissolve with methanol, add kieselguhr and mix thoroughly, extract with the dehydrated alcohol hot reflux, gained extracting solution evaporate to dryness, Radix Astragali total saponins;

Perhaps, with 2) in obtain the Radix Astragali total saponins crude product, use dissolve with methanol, add kieselguhr and mix thoroughly, extract with the dehydrated alcohol hot reflux, gained extracting solution evaporate to dryness, Radix Astragali total saponins;

Perhaps, with 3) in obtain the Radix Astragali total saponins crude product, use water dissolution, last macroporous resin column, water and aquiferous ethanol gradient elution merge the 70%-98% ethanol elution, evaporate to dryness, Radix Astragali total saponins;

Perhaps, with 3) in obtain the Radix Astragali total saponins crude product, use water dissolution, with the saturated n-butanol extraction of NaOH of 0.1N, concentrated extract is to the extractum shape earlier, the reuse water dissolution, last macroporous resin column, water and aquiferous ethanol gradient elution merge the 70%-98% ethanol elution, evaporate to dryness gets Radix Astragali total saponins.

2, according to the described astragaloside of claim 1, it is characterized in that: the model of macroporous resin column is D101, AB-8; The alkaline solution that the n-butyl alcohol that is used to extract is saturated is the NaOH of 0.1N.

3, according to the described astragaloside of claim 1, it is characterized in that: ethanol extraction extractum and diatomaceous weight ratio are 1: 1-3.

4, according to the described astragaloside of claim 3, it is characterized in that: ethanol extraction extractum and diatomaceous weight ratio are for being 1: 2.

5, be used to prevent and treat the pharmaceutical composition of cardiovascular disease, wherein contain the astragaloside and the pharmaceutically acceptable carrier of the claim 1 for the treatment of effective dose.