CN116199732A - Method for increasing leaching of effective components of astragalus membranaceus by utilizing sugar - Google Patents

Abstract

The invention provides a method for increasing leaching of effective components of astragalus by using sugar. The method comprises the following steps: (1) preparing a sugar solution and/or a sugar alcohol solution; (2) Soaking radix astragali in the sugar solution and/or sugar alcohol solution; and (3) heating and extracting the astragalus in the sugar solution and/or the sugar alcohol solution, and filtering to obtain astragalus extract. The method can remarkably increase the extraction rate of the effective components in the astragalus, thereby being beneficial to improving the utilization rate and the curative effect of the astragalus medicinal material.

Description

A method of using sugar to increase the extraction of effective components of Radix Astragali

technical field

The invention relates to the field of extraction of traditional Chinese medicines, in particular to a method for increasing the leaching of effective components of Astragalus membranaceus by using sugar.

Background technique

At present, how to efficiently extract the active ingredients of traditional Chinese medicine and ensure the safety of the extract is an important content in the research field of traditional Chinese medicine extraction. Most of the traditional methods of taking traditional Chinese medicine are decocted in water. Clinical experience and modern research have shown that this method can guarantee the effectiveness and safety of the extract better than organic solvent extraction. Therefore, in the new registration and application of new traditional Chinese medicines released in 2020, it is stipulated that if the declared indications are consistent with the traditional usage, the clinical trials can be moderately reduced or exempted. The Regulations on the Administration of Registration and Approval clarifies the scope of application where clinical trials can be exempted from the development of ancient classic famous prescription compound preparations, including the preparation process is consistent with the records in ancient medical books. Modern research has found that the ethanol extraction efficiency of most components of traditional Chinese medicine is much higher than that of water decoction, but the toxicity of the obtained extract increases. The main reasons for the difference between the two extraction methods are: Chinese medicine contains a wide variety of active ingredients and a wide range of polarity, and ethanol extraction increases the extraction rate of weakly polar and medium polar components, resulting in ethanol extracts that are different from those used in traditional clinical practice. The composition of active ingredients in the decoction is different, so it may have a certain impact on the safety of the extract. In addition, the most commonly used decoction method for traditional Chinese medicine extraction has problems such as low extraction rate and incomplete extraction.

Therefore, how to improve the extraction rate of Chinese medicine extracts on the basis of following the ancient times is an important research direction in the study of Chinese medicine extraction.

Contents of the invention

Based on this, the present invention provides a high-efficiency extraction method of Astragalus decoction pieces, using a certain concentration of sugar water and/or sugar alcohol water instead of water as the extraction solvent, compared with the extraction by traditional decoction method, the extraction rate is significantly improved , thereby improving the utilization rate and curative effect of Astragalus medicinal materials. The sugar used in this study is a common excipient or edible sugar in traditional Chinese medicine, so it is safe and low-priced, and will not affect the safety of the extract.

Specifically, according to one aspect of the present invention, there is provided a method for utilizing sugar to increase the leaching of active ingredients of Radix Astragalus, the method comprising the following steps:

(1) preparing sugar solution and/or sugar alcohol solution;

(2) Soaking Radix Astragali in the sugar solution and/or the sugar alcohol solution; and

(3) Heat and extract the Radix Astragali in the sugar solution and/or the sugar alcohol solution, and obtain an extract of Radix Astragali through filtration.

According to another aspect of the present invention, there is provided a method for utilizing sugar to increase the leaching of active ingredients of Radix Astragali, characterized in that the method comprises the following steps:

(1) preparing sugar solution and/or sugar alcohol solution;

(2) Soaking Radix Astragali in the sugar solution and/or the sugar alcohol solution;

(3) heating and extracting the Astragalus membranaceus in the sugar solution and/or the sugar alcohol solution, and obtaining the first Astragalus membranaceus extract by filtering;

(4) adding the sugar solution and/or the sugar alcohol solution to the Radix Astragali, heating and extracting the Radix Astragali to obtain a second extract of Radix Astragali; and

(5) Combine the first Astragalus extract and the second Astragalus extract to obtain the Astragalus extract (ie, the total Astragalus extract or the third Astragalus extract).

Further, the sugar is selected from one or more of monosaccharides, disaccharides and trisaccharides.

Further, the sugar alcohol is selected from one or more of sorbitol, mannitol, erythritol, maltitol, lactitol and xylitol.

Further, the monosaccharide is selected from one or more of glucose, fructose, galactose, mannose, galactose, sorbose, rhamnose, ribose, xylose and deoxyribose.

Further, the disaccharide is selected from one or more of maltose, sucrose, lactose and trehalose.

Further, the monosaccharide is glucose and/or fructose.

Further, the disaccharide is selected from one or more of maltose, sucrose and trehalose.

Further, the trisaccharide is raffinose.

Further, the disaccharide is sucrose.

Further, in the sugar solution or the sugar alcohol solution, the concentration range of the sugar or the sugar alcohol is 0.1-30g/100mL.

Further, the concentration range of the sugar or the sugar alcohol is 0.25-10g/100mL.

Further, the concentration range of the sugar or the sugar alcohol is 1-10g/100mL.

Further, the concentration range of the sugar or the sugar alcohol is 1-1.25g/100mL or the concentration of the sugar or the sugar alcohol is about 10g/100mL.

Further, the active ingredient contains saponins and/or flavonoids.

Further, the saponins are triterpene saponins.

Further, the saponin compound is astragaloside IV.

Further, the flavonoids are isoflavones.

Further, the flavonoid compound is selected from one or more of acteosin glucoside, formononetin and acteosin.

Further, in step (2), the soaking is soaking at room temperature.

Further, the step of soaking includes soaking the astragalus root in the sugar solution and/or the sugar alcohol solution for 10-120 minutes at room temperature.

Further, the step of soaking includes soaking the astragalus root in the sugar solution and/or the sugar alcohol solution for 20-60 minutes at room temperature.

Further, the step of soaking includes soaking the astragalus in the sugar solution and/or the sugar alcohol solution at room temperature for about 30 minutes.

Further, the weight-to-volume ratio of the astragalus to the sugar solution and/or the sugar alcohol solution is 1:1-1:50.

Further, the weight-to-volume ratio of the astragalus to the sugar solution and/or the sugar alcohol solution is 1:1-1:20.

Further, the weight-to-volume ratio of the astragalus to the sugar solution and/or the sugar alcohol solution is 1:6-1:7.

Further, in step (3), the heating extraction is heating reflux extraction.

Further, the heating time is 20 minutes to 60 minutes.

Further, the heating time is about 30 minutes.

According to another aspect of the present invention, there is provided a use of the above method in the preparation of astragalus extract or astragalus concentrate.

Further, the Astragalus concentrated solution is obtained by concentrating the Astragalus extract obtained in step (5) (ie, the total Astragalus extract or the third Astragalus extract) in a water bath at 20°C to 100°C.

Further, in step (5), the concentration is completed in a water bath at 60°C to 80°C.

Further, the concentration is completed in a water bath at about 70°C.

According to another aspect of the present invention, there is provided a use of the above method in the preparation of a pharmaceutical composition containing astragalus.

According to another aspect of the present invention, there is provided a use of the above method in the preparation of pharmaceutical preparations, functional foods, health foods and other products containing astragalus.

Beneficial effects of the present invention:

Astragalus is a commonly used Chinese medicinal material, which is the root of the leguminous plant Astragalus mongolica. Excavated in spring and autumn, removed the soil, fibrous roots and root heads, sun-dried to 60-70% dry, straightened and bundled, then dried in the sun. Functions and indications are: invigorating qi and solidifying the surface, detoxification and pus drainage, diuresis, and muscle regeneration. The main chemical components of Astragalus are astragalus polysaccharides, saponins, flavonoids and amino acids; its pharmacological effects are to improve immune function, enhance anti-oxidation, anti-radiation and anti-cancer effects, protect cardiovascular and cerebrovascular, liver, kidney and lung functions, protect Brain cells, improve memory, dilate vascular smooth muscle, hormone-like effects, antibacterial and virus-inhibiting effects, lower blood lipids, lower blood sugar, reduce complications of diabetes, etc. Our research has found that, compared with the traditional water decocting method, the method of the present invention can fully improve the extraction rate of the effective components of Astragalus membranaceus.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Apparently, the described embodiments are part of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative efforts belong to the protection scope of the present invention.

It should be noted that, in the case of no conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below in conjunction with examples.

The present invention will be described in further detail below in conjunction with specific examples, and these examples should not be construed as limiting the scope of protection claimed by the present invention.

As described in the background technology section, the alcohol extract is different from the active ingredients in the traditional clinical decoction, so it may have a certain impact on the safety of the alcohol extract, but the extraction efficiency of traditional water decoction is not high. In order to solve the above problems, the present invention provides a method for utilizing sugar to increase the leaching of active ingredients of Radix Astragalus, the method comprising the following steps:

(1) preparing sugar solution and/or sugar alcohol solution;

(2) Soaking Radix Astragali in the sugar solution and/or the sugar alcohol solution; and

(3) Heat and extract the Radix Astragali in the sugar solution and/or the sugar alcohol solution, and obtain an extract of Radix Astragali through filtration.

The ability of sugar solution and/or sugar alcohol solution to improve the extraction rate of active ingredients in Radix Astragali is mainly related to two factors:

(1) Whether the liquid can infiltrate the solid decoction pieces is related to its surface tension. Those with a small surface tension coefficient (about 30×10 ^-3 N/m) can almost wet solids; water has a large surface tension coefficient, and it can only wet certain solids. The surface tension coefficient of sugar aqueous solution is smaller than that of pure water, so the solution can better enter the plant cells and release active ingredients, so it can improve the infiltration of Astragalus decoction pieces and the dissolution of active ingredients;

(2) The sugar can form hydrogen bonds and other forces between the active ingredients in the Astragalus decoction pieces, thus improving the extraction efficiency of the Astragalus extract components;

Therefore, compared with water decoction, this method can fully improve the extraction rate of effective components of Astragalus membranaceus.

In the actual working process, the number of extractions of the alcohol and/or sugar alcohol solution can be determined according to actual needs, which can be one time or several times, such as 2-10 times.

According to another aspect of the present invention, there is provided a method for utilizing sugar to increase the leaching of active ingredients of Radix Astragali, characterized in that the method comprises the following steps:

(1) preparing sugar solution and/or sugar alcohol solution;

(2) Soaking Radix Astragali in the sugar solution and/or the sugar alcohol solution;

(3) heating and extracting the Astragalus membranaceus in the sugar solution and/or the sugar alcohol solution, and obtaining the first Astragalus membranaceus extract by filtering;

(4) adding the sugar solution and/or the sugar alcohol solution to the Radix Astragali, heating and extracting the Radix Astragali to obtain a second extract of Radix Astragali; and

(5) Combine the first Astragalus extract and the second Astragalus extract to obtain the Astragalus extract (ie, the total Astragalus extract or the third Astragalus extract).

The term "about" or "approximately" with respect to a numerical value means ± 5% of the numerical value, but expressly includes the exact numerical value. For example, a concentration of "about" 0.5 g crude drug/mL refers to a concentration of from 0.475 g crude drug/mL to 0.525 g crude drug/mL, but specifically includes concentrations of exactly 0.5 g crude drug/mL.

In a preferred embodiment, the sugar is selected from one or more of monosaccharides, disaccharides and trisaccharides.

In a preferred embodiment, the sugar alcohol is selected from one or more of sorbitol, mannitol, erythritol, maltitol, lactitol and xylitol. During practical application, the above-mentioned sugar alcohols can be replaced with other sugar alcohols in the prior art according to the actual situation.

In a preferred embodiment, the monosaccharide is selected from one or more of glucose, fructose, galactose, mannose, galactose, sorbose, rhamnose, ribose, xylose and deoxyribose. During practical application, the above-mentioned monosaccharides can be replaced with other monosaccharides in the prior art according to the actual situation.

In a preferred embodiment, the disaccharide is selected from one or more of maltose, sucrose, lactose and trehalose. During practical application, the above-mentioned disaccharides can be replaced with other disaccharides in the prior art according to the actual situation.

In a preferred embodiment, the monosaccharide is glucose and/or fructose.

In a preferred embodiment, the disaccharide is selected from one or more of maltose, sucrose and trehalose.

In a preferred embodiment, the trisaccharide is raffinose.

In order to further increase the extraction rate of active ingredients in Radix Astragali, in a preferred embodiment, the disaccharide is sucrose.

In a preferred embodiment, in the sugar solution or the sugar alcohol solution, the concentration of the sugar or the sugar alcohol is in the range of 0.1-30g/100mL.

In a preferred embodiment, the concentration range of the sugar or the sugar alcohol is 0.25-10 g/100mL.

In a preferred embodiment, the concentration of the sugar or the sugar alcohol is in the range of 1-10 g/100 mL.

In order to further improve the extraction rate of active ingredients in Radix Astragali, in a preferred embodiment, the concentration range of the sugar or the sugar alcohol is 1-1.25g/100mL or the concentration of the sugar or the sugar alcohol is about 10g/100mL .

The term "about" or "approximately" with respect to a numerical value means ± 5% of the numerical value, but expressly includes the exact numerical value. For example, a concentration of "about" 10 g/100 mL refers to a concentration of from 9.5 g/100 mL to 10.5 g/100 mL, but expressly includes concentrations of exactly 10 g/100 mL.

In a preferred embodiment, the active ingredient contains saponins and/or flavonoids.

In a preferred embodiment, the saponins are triterpene saponins. In practical application, the saponins include other saponins that have been identified from Radix Astragali in the prior art except the triterpene saponins.

In a preferred embodiment, the saponin compound is astragaloside IV.

In a preferred embodiment, the flavonoids are isoflavones.

In practical application, the flavonoids include other flavonoids that have been identified from Radix Astragalus in the prior art except the isoflavones.

In a preferred embodiment, the flavonoid compound is selected from one or more of acteosin glucoside, formononetin and actecoisoflavone.

In a preferred embodiment, in step (2), the soaking is soaking at room temperature.

In a preferred embodiment, the soaking step comprises soaking the astragalus root in the sugar solution and/or the sugar alcohol solution for 10-120 minutes at room temperature.

In a preferred embodiment, the step of soaking comprises soaking the astragalus in the sugar solution and/or the sugar alcohol solution for 20-60 minutes at room temperature.

In a preferred embodiment, the step of soaking includes soaking the astragalus in the sugar solution and/or the sugar alcohol solution at room temperature for about 30 minutes.

The term "about" or "approximately" with respect to a numerical value means ± 5% of the numerical value, but expressly includes the exact numerical value. For example, a time of "about" 30 min refers to a time from 28.5 min to 31.5 min, but also expressly includes a time of exactly 30 min.

In a preferred embodiment, the weight-to-volume ratio of the astragalus to the sugar solution and/or the sugar alcohol solution is 1:1˜1:50.

In a preferred embodiment, the weight-to-volume ratio of the astragalus to the sugar solution and/or the sugar alcohol solution is 1:1˜1:20.

In a preferred embodiment, the weight-to-volume ratio of the astragalus to the sugar solution and/or the sugar alcohol solution is 1:6˜1:7.

In a preferred embodiment, in step (3), the heating extraction is heating reflux extraction.

In a preferred embodiment, the heating time is 20 minutes to 60 minutes.

In a preferred embodiment, the heating time is about 30 minutes.

The term "about" or "approximately" with respect to a numerical value means ± 5% of the numerical value, but expressly includes the exact numerical value. For example, a time of "about" 30 min refers to a time from 28.5 min to 31.5 min, but also expressly includes a time of exactly 30 min.

According to another aspect of the present invention, there is provided a use of the above method in the preparation of astragalus extract or astragalus concentrate.

In a preferred embodiment, the Astragalus concentrated solution is obtained by concentrating the Astragalus extract obtained in step (5) (ie the total Astragalus extract or the third Astragalus extract) in a water bath at 20°C to 100°C.

In a preferred embodiment, in step (5), the concentration is completed in a water bath at 60°C to 80°C.

In a preferred embodiment, the concentration is accomplished in a water bath at about 70°C.

The term "about" or "approximately" with respect to a numerical value means ± 5% of the numerical value, but expressly includes the exact numerical value. For example, a temperature of "about" 70°C refers to a scale from 66.5°C to 73.5°C, but also expressly includes a temperature of exactly 70°C.

According to another aspect of the present invention, there is provided a use of the above method in the preparation of a pharmaceutical composition containing astragalus.

According to another aspect of the present invention, there is provided a use of the above method in the preparation of pharmaceutical preparations, functional foods, health foods and other products containing astragalus.

Among them, functional foods are foods that can be convincingly proven to be beneficial to one or more functions of the body, have sufficient nutritional effects to improve health status, or reduce illness.

Among them, health food refers to food that claims to have specific health functions or to supplement vitamins and minerals, that is, it is suitable for consumption by specific groups of people, has the function of regulating the body, is not for the purpose of treating diseases, and does not cause any acute, sub-standard effects on the human body. Acute or chronic hazard food.

Wherein other products refer to all products included in the prior art except pharmaceutical preparations, functional foods and health foods containing astragalus, including liquid and solid forms.

Example 1 Experiment of Extracting Raw Astragalus Pieces with Different Sugar Solutions

1.1 Preparation method of different sugar-Astragalus extract (extract twice with different 1% sugar)

The same batch of raw Astragalus decoction pieces (from Hunyuan, Shanxi) took 5 parts (HQ, HQ+1% sucrose, HQ+1% glucose, HQ+1% fructose, HQ+1% trehalose), 50g each. Add 7 times the amount of water (350ml water), 1% aqueous glucose solution (3.5g glucose and water to 350ml), 1% fructose aqueous solution (3.5g fructose and water to 350ml), 1% sucrose aqueous solution (3.5g sucrose Add water to 350ml), 1% trehalose aqueous solution (3.5g trehalose, add water to 350ml), soak for 30min, reflux for 30min, filter with gauze while it is hot, add 6 times the amount of water (300ml water) to the dregs, 1% aqueous glucose solution (3.0g glucose with water to 300ml), 1% fructose aqueous solution (3.0g fructose with water to 300ml), 1% sucrose aqueous solution (3.0g sucrose with water to 300ml), 1% trehalose aqueous solution (Add 3.0 g of trehalose to 300 ml of water), reflux for 20 minutes, filter while hot, and combine the two filtrates to obtain different sugars-extracts of Astragalus membranaceus.

1.2 Determination of astragaloside IV, acteosin glucoside, formononetin and acteosin

1.2.1 Instruments and reagents

Agilent 1260 high performance liquid chromatograph equipped with DAD detector (Agilent Corporation, USA); Agilent 1200 high performance liquid chromatography instrument equipped with ELSD detector (Agilent Corporation USA); DZKW-4 electronic constant temperature water bath (Beijing ZTE Weiye Instrument Co., Ltd.); DK-98-IIA electric heating constant temperature water bath (Tianjin Test Instrument Co., Ltd.); ME204/02 electronic balance [Mettler-Toledo Instrument (Shanghai) Co., Ltd., parts per 100,000 One day level]; KQ-250DE type numerical control ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); HC-3018 high-speed centrifuge (Anhui Zhongke Zhongjia Scientific Instrument Co., Ltd.); solid phase extraction column UniEIutC18EC column (1000mg/6ml ) (China Spectrum Instrument Beijing Technology Co., Ltd.).

The reference substance astragaloside (batch number CHB170727), calycosin glucoside (batch number CHB161105), formononetin (batch number CHB150517), and acteocoside (batch number CHB161104) were all purchased from Chengdu Croma Biotechnology Co., Ltd., with a purity of HPlC≥ 98%; Acetonitrile (chromatographically pure, Thermo Fisher Scientific Co., Ltd.), methanol (chromatographically pure, Thermo Fisher Scientific Co., Ltd.), formic acid is analytically pure, purchased from Sinopharm Chemical Reagent Co., Ltd.; water is Wahaha pure water.

1.2.2 Preparation of the test solution

The preparation of saponins component need testing solution: get different sugar-astragali extract 5ml, add 20ml60% methyl alcohol, ultrasonically mix, add 10ml ammoniacal liquor, mix well, be placed in 3600r/min centrifugal 10min, supernatant passes Chinese spectrum UniEIut C18 EC column (1000mg/6ml), elute with 10ml of pure water, then 5ml of methanol, collect the methanol eluate, centrifuge at 8000r/min for 5min, take the supernatant, pass through a 0.45μm microporous membrane to obtain the product.

Preparation of the test solution of flavonoid components: Take 300 μl of different sugar-astragalus extracts, add 700 μl of water, put them in a 1.5ml centrifuge tube, centrifuge at 12000r/min for 5min, take the supernatant, and pass through a 0.45μm microporous membrane to obtain the product.

1.2.3 Preparation of reference solution

Take astragaloside IV, acteosin glucoside, formononetin and acteosin reference substance appropriate amount, accurately weighed, add methanol to prepare 1mg/ml stock solution. Dilute the stock solution into different serial concentrations to establish a working curve.

The linear equation of astragaloside IV is lnY=1.3271lnX+9.9314, r=1.0000; the linear equation of actecoside glucoside is Y=35129X+14, r=0.9998; the linear equation of formononetin is Y=10024X+33, r=0.9998; the linear equation of calycosin is Y=4656X+25, r=0.9995.

1.2.4 Content determination method

Determination method of saponins content: adopt Agilent 1200 type liquid chromatograph (equipped with ELSD detector) to analyze, chromatographic column: Thermo Acclaim 120 C18 (250 * 4.6mm, 5 μ m); Mobile phase: 0.1% formic acid water (A )～0.1% formic acid acetonitrile (B), gradient elution: 0～5min, 5%～10%B; 5～10min, 10%～32%B; 10～30min, 32%～45%B; 30～35min , 45%～95%B; 35～40min, 95%～20%B), column temperature: room temperature, flow rate 1ml/min, drift tube temperature 100°C, carrier gas flow rate 2.5l/min, injection volume 20μl.

Flavonoid content determination method: adopt Agilent 1260 type high performance liquid chromatograph (equipped with DAD detector) to analyze, chromatographic column: Thermo Acclaim 120 C18 (250 * 4.6mm, 5 μ m); Mobile phase: 0.1% formic acid aqueous solution ( A) with 0.1% formic acid acetonitrile (B), gradient elution: 0~8min, 5%~20%B; 8~15min, 20%~25%B; 15~20min, 25%B; 20~30min, 25% % ~ 40% B; 30 ~ 40min, 40% ~ 60% B; flow rate 1ml/min; detection wavelength 260nm; column temperature 25 ℃; injection volume 10μl.

1.3 Experimental results

Table 1 shows the content determination results of active ingredients in different sugar-astragalus extracts.

Table 1 Determination results of active ingredients in different sugar-astragalus extracts

*P<0.05 VS water

As can be seen from Table 1, sucrose, glucose, fructose, and trehalose (1g/100ml concentration) can all increase the content of active ingredients in Astragalus membranaceus to varying degrees, and the effects of sucrose and trehalose are the most obvious, but the ratio of sucrose to seaweed Sugar is more economical.

Example 2 Experiment of Extracting Raw Astragalus Pieces with Different Sugar Solutions

2.1 Preparation method of different sugar-Astragalus extract (extract once with different 1% sugar)

The same batch of raw Astragalus decoction pieces (from Hunyuan, Shanxi) took 4 parts (HQ, HQ+1% glucose, HQ+1% sucrose, HQ+1% maltose), each 50g. Add 7 times the amount of water (350ml water), 1% glucose aqueous solution (3.5g glucose is added to 350ml with water), 1% sucrose aqueous solution (3.5g sucrose is added to 350ml with water), 1% maltose aqueous solution (3.5g maltose Add water to make the volume 350ml), soak for 30min, reflux for 30min, filter with gauze while it is hot, and obtain different sugar-astragalus extracts.

2.2 Determination of astragaloside IV, acteosin glucoside, formononetin and acteosin

2.2.1 Instruments and reagents

Agilent 1260 high performance liquid chromatograph equipped with DAD detector (Agilent Corporation, USA); Agilent 1200 high performance liquid chromatography instrument equipped with ELSD detector (Agilent Corporation USA); DZKW-4 electronic constant temperature water bath (Beijing ZTE Weiye Instrument Co., Ltd.); DK-98-IIA electric heating constant temperature water bath (Tianjin Test Instrument Co., Ltd.); ME204/02 electronic balance [Mettler-Toledo Instrument (Shanghai) Co., Ltd., parts per 100,000 One day level]; KQ-250DE type numerical control ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); HC-3018 high-speed centrifuge (Anhui Zhongke Zhongjia Scientific Instrument Co., Ltd.); solid phase extraction column UniEIut C18 EC column (1000mg /6ml) (China Spectrum Instrument Beijing Technology Co., Ltd.).

The reference substance astragaloside (batch number CHB170727), calycosin glucoside (batch number CHB161105), formononetin (batch number CHB150517), and acteocoside (batch number CHB161104) were all purchased from Chengdu Croma Biotechnology Co., Ltd., with a purity of HPlC≥ 98%; acetonitrile (chromatographically pure, Thermo Fisher Scientific Co., Ltd.), methanol (chromatographically pure, Thermo Fisher Scientific Co., Ltd.), and formic acid were analytically pure, purchased from Sinopharm Chemical Reagent Co., Ltd. The water is Wahaha purified water.

2.2.2 Preparation of the test solution

The preparation of saponins component need testing solution: get different sugar-astragali extract 5ml, add 20ml60% methyl alcohol, ultrasonically mix, add 10ml ammoniacal liquor, mix well, be placed in 3600r/min centrifugal 10min, supernatant passes Chinese spectrum UniEIut C18 EC column (1000mg/6ml), elute with 10ml of pure water, then 5ml of methanol, collect the methanol eluate, centrifuge at 8000r/min for 5min, take the supernatant, pass through a 0.45μm microporous membrane to obtain the product.

Preparation of the test solution of flavonoid components: Take 300 μl of different sugar-astragalus extracts, add 700 μl of water, put them in a 1.5ml centrifuge tube, centrifuge at 12000r/min for 5min, take the supernatant, and pass through a 0.45μm microporous membrane to obtain the product.

2.2.3 Preparation of reference solution

Take astragaloside IV, acteosin glucoside, formononetin and acteosin reference substance appropriate amount, accurately weighed, add methanol to prepare 1mg/ml stock solution. Dilute the stock solution into different serial concentrations to establish a working curve.

The linear equation of astragaloside IV is lnY=1.3271lnX+9.9314, r=1.0000; the linear equation of actecoside glucoside is Y=35129X+14, r=0.9998; the linear equation of formononetin is Y=10024X+33, r=0.9998; the linear equation of calycosin is Y=4656X+25, r=0.9995.

2.2.4 Content determination method

Determination method of saponins content: adopt Agilent 1200 type liquid chromatograph (equipped with ELSD detector) to analyze, chromatographic column: Thermo Acclaim 120 C18 (250 * 4.6mm, 5 μ m); Mobile phase: 0.1% formic acid water (A )～0.1% formic acid acetonitrile (B), gradient elution: 0～5min, 5%～10%B; 5～10min, 10%～32%B; 10～30min, 32%～45%B; 30～35min , 45%～95%B; 35～40min, 95%～20%B), column temperature: room temperature, flow rate 1ml/min, drift tube temperature 100°C, carrier gas flow rate 2.5l/min, injection volume 20μl.

Flavonoid content determination method: adopt Agilent 1260 type high performance liquid chromatograph (equipped with DAD detector) to analyze, chromatographic column: Thermo Acclaim 120 C18 (250 * 4.6mm, 5 μ m); Mobile phase: 0.1% formic acid aqueous solution ( A) with 0.1% formic acid acetonitrile (B), gradient elution: 0~8min, 5%~20%B; 8~15min, 20%~25%B; 15~20min, 25%B; 20~30min, 25% % ~ 40% B; 30 ~ 40min, 40% ~ 60% B; flow rate 1ml/min; detection wavelength 260nm; column temperature 25 ℃; injection volume 10μl.

2.3 Experimental results

Table 2 shows the content determination results of active ingredients in different sugar-astragalus extracts.

Table 2 Determination results of active ingredients in different sugar-Astragalus extracts

*P<0.05 VS water

It can be seen from Table 2 that glucose, sucrose, and maltose (1g/100ml concentration) can all increase the content of active ingredients in Radix Astragali to varying degrees, and the effect of sucrose is the most obvious.

Example 3 Extraction of Raw Astragalus Pieces with Different Sugar Solutions

3.1 Preparation method of different sugar-Astragalus extract (extract twice with different 1% sugar)

The same batch of raw Astragalus decoction pieces (from Lingchuan, Shanxi) took 4 parts (HQ, HQ+1% sucrose, HQ+1% maltose, HQ+1% lactose), each 50g. Add 7 times the amount of water (350ml water), 1% sucrose aqueous solution (3.5g sucrose, add water to 350ml), 1% maltose aqueous solution (3.5g maltose, add water to 350ml), 1% lactose aqueous solution (3.5g lactose Add water to 350ml), soak for 30min, reflux for 30min, filter with gauze while hot, add 6 times the amount of water (300ml water), 1% sucrose aqueous solution (3.0g sucrose and water to 300ml), 1% maltose Aqueous solution (3.0g maltose, add water to 300ml), 1% lactose aqueous solution (3.0g lactose, add water to 300ml), reflux for 20min, filter while hot, combine 2 filtrates to obtain different sugars - astragalus extract.

3.2 Determination of astragaloside IV, acteosin glucoside, formononetin and acteosin

3.2.1 Instruments and reagents

Agilent 1260 high performance liquid chromatograph equipped with DAD detector (Agilent Corporation, USA); Agilent 1200 high performance liquid chromatography instrument equipped with ELSD detector (Agilent Corporation USA); DZKW-4 electronic constant temperature water bath (Beijing ZTE Weiye Instrument Co., Ltd.); DK-98-IIA electric heating constant temperature water bath (Tianjin Test Instrument Co., Ltd.); ME204/02 electronic balance [Mettler-Toledo Instrument (Shanghai) Co., Ltd., parts per 100,000 One day level]; KQ-250DE type numerical control ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); HC-3018 high-speed centrifuge (Anhui Zhongke Zhongjia Scientific Instrument Co., Ltd.); solid phase extraction column UniEIutC18EC column (1000mg/6ml ) (China Spectrum Instrument Beijing Technology Co., Ltd.).

The reference substance astragaloside (batch number CHB170727), calycosin glucoside (batch number CHB161105), formononetin (batch number CHB150517), and acteocoside (batch number CHB161104) were all purchased from Chengdu Croma Biotechnology Co., Ltd., with a purity of HPlC≥ 98%; acetonitrile (chromatographically pure, Thermo Fisher Scientific Co., Ltd.), methanol (chromatographically pure, Thermo Fisher Scientific Co., Ltd.), and formic acid were analytically pure, purchased from Sinopharm Chemical Reagent Co., Ltd. The water is Wahaha purified water.

3.2.2 Preparation of the test solution

The preparation of saponins component need testing solution: get different sugar-astragali extract 5ml, add 20ml 60% methyl alcohol, ultrasonically mix, add 10ml ammoniacal liquor, mix well, be placed in 3600r/min centrifugal 10min, supernatant is crossed Chinese spectrum UniEIut C18 EC column (1000mg/6ml), elute with 10ml of pure water, then 5ml of methanol, collect the methanol eluate, centrifuge at 8000r/min for 5min, take the supernatant, and pass through a 0.45μm microporous membrane to obtain .

Preparation of the test solution of flavonoid components: Take 300 μl of different sugar-astragalus extracts, add 700 μl of water, put them in a 1.5ml centrifuge tube, centrifuge at 12000r/min for 5min, take the supernatant, and pass through a 0.45μm microporous membrane to obtain the product.

3.2.3 Preparation of reference solution

Take astragaloside IV, acteosin glucoside, formononetin and acteosin reference substance appropriate amount, accurately weighed, add methanol to prepare 1mg/ml stock solution. Dilute the stock solution into different serial concentrations to establish a working curve.

The linear equation of astragaloside IV is lnY=1.3271lnX+9.9314, r=1.0000; the linear equation of actecoside glucoside is Y=35129X+14, r=0.9998; the linear equation of formononetin is Y=10024X+33, r=0.9998; the linear equation of calycosin is Y=4656X+25, r=0.9995.

3.2.4 Content determination method

Determination method of saponins content: adopt Agilent 1200 type liquid chromatograph (equipped with ELSD detector) to analyze, chromatographic column: Thermo Acclaim 120 C18 (250 * 4.6mm, 5 μ m); Mobile phase: 0.1% formic acid water (A )～0.1% formic acid acetonitrile (B), gradient elution: 0～5min, 5%～10%B; 5～10min, 10%～32%B; 10～30min, 32%～45%B; 30～35min , 45%～95%B; 35～40min, 95%～20%B), column temperature: room temperature, flow rate 1ml/min, drift tube temperature 100°C, carrier gas flow rate 2.5l/min, injection volume 20μl.

Flavonoid content determination method: adopt Agilent 1260 type high performance liquid chromatograph (equipped with DAD detector) to analyze, chromatographic column: Thermo Acclaim 120 C18 (250 * 4.6mm, 5 μ m); Mobile phase: 0.1% formic acid aqueous solution ( A) with 0.1% formic acid acetonitrile (B), gradient elution: 0~8min, 5%~20%B; 8~15min, 20%~25%B; 15~20min, 25%B; 20~30min, 25% % ~ 40% B; 30 ~ 40min, 40% ~ 60% B; flow rate 1ml/min; detection wavelength 260nm; column temperature 25 ℃; injection volume 10μl.

3.3 Experimental results

Table 3 shows the content determination results of active ingredients in different sugar-astragalus extracts.

Table 3 Determination results of active ingredients in different sugar-Astragalus extract

*P<0.05 VS water

It can be seen from Table 3 that sucrose, maltose, and lactose (1g/100ml concentration) can all increase the content of active ingredients in Astragalus membranaceus to varying degrees, and sucrose has the most obvious effect.

Based on the results in Table 1 and Table 3, it can be seen that sucrose can significantly increase the extraction rate of the main active components in Astragalus membranaceus, including flavonoids (actecoisoflavone glucoside, formononetin, and actecoside) and saponins (astragaloside IV).

Example 4 Extraction of Raw Astragalus Pieces with Sucrose Solutions of Different Concentrations

4.1 Preparation of different concentrations of sucrose-Astragalus extract (0-10% different concentrations of sucrose extraction)

The same batch of raw Astragalus decoction pieces (from Lingchuan, Shanxi) took 6 parts (HQ, HQ+0.25% sucrose, HQ+0.5% sucrose, HQ+1% sucrose, HQ+2% sucrose, HQ+10% sucrose), 50g per serving. Add 7 times the amount of water (350ml water), 0.25% sucrose aqueous solution (0.9g sucrose, add water to 350ml), 0.5% sucrose aqueous solution (1.8g sucrose, add water to 350ml), 1% sucrose aqueous solution (3.5g sucrose Add water to 350ml), 1.25% sucrose aqueous solution (4.4g sucrose, add water to 350ml), 1.5% sucrose aqueous solution (5.3g sucrose to 350ml), 2% sucrose aqueous solution (7g sucrose to 350ml) , 10% sucrose aqueous solution (35g sucrose, add water to make up to 350ml), soak for 30min, reflux for 30min, filter with gauze while hot, then add 6 times the amount of water (300ml water), 0.25% sucrose aqueous solution (0.8g sucrose add water 0.5% sucrose aqueous solution (1.5g sucrose, add water to 300ml), 1% sucrose aqueous solution (3.0g sucrose, add water to 300ml), 1.25% sucrose aqueous solution (3.8g sucrose, add water to 300ml) , 1.5% sucrose aqueous solution (4.5g sucrose, add water to 300ml), 2% sucrose aqueous solution (6.0g sucrose, add water to 300ml), 10% sucrose aqueous solution (30g sucrose, add water to 300ml), reflux for 20min, while hot Filtrate and combine the two filtrates to obtain extracts of Astragalus membranaceus with different concentrations of sucrose.

4.2 Determination of astragaloside IV, acteosin glucoside, formononetin and acteosin

4.2.1 Instruments and reagents

Agilent 1260 high performance liquid chromatograph equipped with DAD detector (Agilent Corporation, USA); Agilent 1200 high performance liquid chromatography instrument equipped with ELSD detector (Agilent Corporation USA); DZKW-4 electronic constant temperature water bath (Beijing ZTE Weiye Instrument Co., Ltd.); DK-98-IIA electric heating constant temperature water bath (Tianjin Test Instrument Co., Ltd.); ME204/02 electronic balance [Mettler-Toledo Instrument (Shanghai) Co., Ltd., parts per 100,000 One day level]; KQ-250DE type numerical control ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); HC-3018 high-speed centrifuge (Anhui Zhongke Zhongjia Scientific Instrument Co., Ltd.); solid phase extraction column UniEIutC18EC column (1000mg/6ml ) (China Spectrum Instrument Beijing Technology Co., Ltd.).

The reference substance astragaloside (batch number CHB170727), calycosin glucoside (batch number CHB161105), formononetin (batch number CHB150517), and acteocoside (batch number CHB161104) were all purchased from Chengdu Croma Biotechnology Co., Ltd., with a purity of HPlC≥ 98%; acetonitrile (chromatographically pure, Thermo Fisher Scientific Co., Ltd.), methanol (chromatographically pure, Thermo Fisher Scientific Co., Ltd.), and formic acid were analytically pure, purchased from Sinopharm Chemical Reagent Co., Ltd. The water is Wahaha purified water.

4.2.2 Preparation of the test solution

The preparation of saponins component need testing solution: get different sugar-astragali extract 5ml, add 20ml60% methyl alcohol, ultrasonically mix, add 10ml ammoniacal liquor, mix evenly, be placed in 3600r/min centrifugation 10min, supernatant is crossed Chinese spectrum UniEIutC18EC column (1000mg/6ml), eluted with 10ml of pure water, then 5ml of methanol, collected the methanol eluate, centrifuged at 8000r/min for 5min, took the supernatant, passed through a 0.45μm microporous membrane, and obtained.

Preparation of the test solution of flavonoid components: Take 300 μl of different sugar-astragalus extracts, add 700 μl of water, put them in a 1.5ml centrifuge tube, centrifuge at 12000r/min for 5min, take the supernatant, and pass through a 0.45μm microporous membrane to obtain the product.

4.2.3 Preparation of reference solution

Take astragaloside IV, acteosin glucoside, formononetin and acteosin reference substance appropriate amount, accurately weighed, add methanol to prepare 1mg/ml stock solution. Dilute the stock solution into different serial concentrations to establish a working curve.

The linear equation of astragaloside IV is lnY=1.3271lnX+9.9314, r=1.0000; the linear equation of actecoside glucoside is Y=35129X+14, r=0.9998; the linear equation of formononetin is Y=10024X+33, r=0.9998; the linear equation of calycosin is Y=4656X+25, r=0.9995.

4.2.4 Content determination method

Determination method of saponins content: adopt Agilent 1200 type liquid chromatograph (equipped with ELSD detector) to analyze, chromatographic column: Thermo Acclaim 120 C18 (250 * 4.6mm, 5 μ m); Mobile phase: 0.1% formic acid water (A )～0.1% formic acid acetonitrile (B), gradient elution: 0～5min, 5%～10%B; 5～10min, 10%～32%B; 10～30min, 32%～45%B; 30～35min , 45%～95%B; 35～40min, 95%～20%B), column temperature: room temperature, flow rate 1ml/min, drift tube temperature 100°C, carrier gas flow rate 2.5l/min, injection volume 20μl.

Flavonoid content determination method: adopt Agilent 1260 type high performance liquid chromatograph (equipped with DAD detector) to analyze, chromatographic column: Thermo Acclaim 120 C18 (250 * 4.6mm, 5 μ m); Mobile phase: 0.1% formic acid aqueous solution ( A) with 0.1% formic acid acetonitrile (B), gradient elution: 0~8min, 5%~20%B; 8~15min, 20%~25%B; 15~20min, 25%B; 20~30min, 25% % ~ 40% B; 30 ~ 40min, 40% ~ 60% B; flow rate 1ml/min; detection wavelength 260nm; column temperature 25 ℃; injection volume 10μl.

4.3 Experimental results

Table 4 shows the content determination results of active ingredients in the Astragalus extract obtained by extracting raw Astragalus decoction pieces with different concentrations of sucrose solutions.

Table 4 Determination of active ingredient content in the astragalus extract obtained by different concentrations of sucrose

*P<0.05 VS water

It can be seen from Table 4 that the concentration of sucrose in the range of 0.25g/100ml to 1.25g/100ml can increase the content of active ingredients in Radix Astragali to varying degrees, among which the concentration of sucrose in the range of 1g/100ml to 1.25g/100ml The inner effect is most obvious. In addition, when the concentration of sucrose is 10g/100ml, it can also increase the content of active ingredients in Astragalus membranaceus.

What is described above is only an embodiment of the present invention, and common knowledge such as specific structures and characteristics known in the scheme are not described here too much. It should be pointed out that for those skilled in the art, without departing from the premise of the present invention, some modifications and improvements can also be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the availability of patents. The scope of protection required by the present invention shall be based on the content of the claims, and the specific implementation methods and other records in the specification may be used to interpret the content of the claims.

The embodiments of the present invention have been described in detail above, and specific examples are used in this paper to illustrate the principle and implementation of the present invention. The descriptions of the above embodiments are only used to help understand the method and core idea of the present invention. At the same time, any changes or deformations made by those skilled in the art based on the idea of the present invention, based on the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the contents of this specification should not be construed as limiting the present invention.

Claims (9)

1. A method for increasing leaching of active ingredients of astragalus using sugar, the method comprising the steps of:

(1) Preparing a sugar solution and/or a sugar alcohol solution;

(2) Soaking radix astragali in the sugar solution and/or sugar alcohol solution; and

(3) Heating and extracting the astragalus in the sugar solution and/or the sugar alcohol solution, and filtering to obtain astragalus extract.

2. A method for increasing leaching of active ingredients of astragalus using sugar, the method comprising the steps of:

(1) Preparing a sugar solution and/or a sugar alcohol solution;

(2) Soaking radix astragali in the sugar solution and/or sugar alcohol solution;

(3) Heating and extracting the astragalus in the sugar solution and/or the sugar alcohol solution, and filtering to obtain a first astragalus extract;

(4) Adding the sugar solution and/or the sugar alcohol solution into the astragalus, and heating and extracting the astragalus to obtain a second astragalus extract; and

(5) And combining the first astragalus extract and the second astragalus extract to obtain the astragalus extract.

3. The method according to claim 1 or 2, wherein the sugar is selected from one or more of monosaccharides, disaccharides and trisaccharides;

preferably, the sugar alcohol is selected from one or more of sorbitol, mannitol, erythritol, maltitol, lactitol and xylitol;

preferably, the monosaccharide is selected from one or more of glucose, fructose, galactose, mannose, galactose, sorbose, rhamnose, ribose, xylose and deoxyribose;

still preferably, the disaccharide is selected from one or more of maltose, sucrose, lactose and trehalose;

particularly preferably, the monosaccharide is glucose and/or fructose;

still more particularly preferably, the disaccharide is selected from one or more of maltose, sucrose and trehalose;

particularly preferably, the trisaccharide is raffinose;

most preferably, the disaccharide is sucrose.

4. The method according to claim 1 or 2, characterized in that in the sugar solution or the sugar alcohol solution, the concentration of the sugar or the sugar alcohol ranges from 0.1 to 30g/100mL;

preferably, the concentration of the sugar or sugar alcohol ranges from 0.25 to 10g/100mL;

more preferably, the concentration of the sugar or sugar alcohol ranges from 1 to 10g/100mL;

particularly preferably, the concentration of the sugar or the sugar alcohol is in the range of 1-1.25g/100mL or the concentration of the sugar or the sugar alcohol is about 10g/100mL.

5. The method according to claim 1 or 2, wherein the active ingredient comprises saponins and/or flavonoids;

preferably, the saponins compound is a triterpenoid saponins compound;

more preferably, the saponin compound is astragaloside IV;

preferably, the flavonoid compound is an isoflavone compound;

still more particularly preferably, the flavonoid is selected from one or more of calycosin glucoside, formononetin and calycosin.

6. The method according to claim 1 or 2, wherein in step (2),

the soaking is performed at room temperature;

preferably, the step of soaking comprises soaking the astragalus in the sugar solution and/or the sugar alcohol solution at room temperature for 10-120min;

still preferably, the step of soaking includes soaking the astragalus in the sugar solution and/or the sugar alcohol solution at room temperature for 20-60min;

more preferably, the step of soaking comprises soaking the astragalus in the sugar solution and/or the sugar alcohol solution at room temperature for about 30min;

particularly preferably, the weight-to-volume ratio of the astragalus to the sugar solution and/or the sugar alcohol solution is 1:1-1:50;

still more preferably, the weight-to-volume ratio of the astragalus to the sugar solution and/or the sugar alcohol solution is 1:1-1:20;

particularly preferably, the weight-to-volume ratio of the astragalus to the sugar solution and/or the sugar alcohol solution is 1:6-1:7;

still more preferably, in step (3), the heat extraction is heat reflux extraction;

particularly preferably, the heating time is from 20min to 60min;

most preferably, the heating time is about 30 minutes.

7. Use of the method according to any one of claims 1 to 6 for the preparation of an astragalus extract or an astragalus concentrate.

8. Use of a method according to any one of claims 1 to 6 for the preparation of a pharmaceutical composition comprising astragalus.

9. Use of the method according to any one of claims 1 to 6 for the preparation of a pharmaceutical formulation or a functional or health food containing astragalus.