Method for extracting flatstem milkvetch seed glycoside from flatstem milkvetch seed

Abstract

The invention discloses a method for extracting flatstem milkvetch seed glycoside from flatstem milkvetch seed, which comprises the following steps: co-melting a hydrogen bond acceptor and a hydrogen bond donor to obtain DES; the method comprises the following steps: preparing DES and water into an extraction solvent, uniformly mixing flatstem milkvetch seed powder with the extraction solvent, carrying out ultrasonic extraction to obtain an extraction solution of flatstem milkvetch seed glycoside, loading the extraction solution to macroporous adsorption resin, sequentially eluting with distilled water, 50% ethanol, 70% ethanol and 90% ethanol, collecting 70% ethanol eluent, and carrying out vacuum concentration to obtain a crude product of flatstem milkvetch seed glycoside; recrystallizing the flatstem milkvetch seed glycoside crude product to obtain a flatstem milkvetch seed glycoside pure product; after water in the loading effluent of the macroporous adsorption resin is evaporated, the macroporous adsorption resin is reused for extracting flatstem milkvetch seed glycoside, and can be reused for at least three times, so that reuse of the eutectic solvent is realized. According to the method, the flatstem milkvetch seed glycoside can be simply, conveniently and efficiently obtained from the flatstem milkvetch seed, the yield of the extracted flatstem milkvetch seed glycoside is more than 10 times of the extraction rate of a traditional organic solvent, and the method has the advantages of simplicity and convenience in operation, safety, rapidness, low cost and the like, and can realize industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product extraction and separation, and specifically relates to a method for obtaining complanatus glycoside from complanatus complanatus by utilizing DES combined with macroporous adsorption resin chromatography column technology. Background Art

[0002] Astragalus complanatus is the dried mature seed of the leguminous plant Astragalus complanatus R.Br. It is a traditional Chinese medicine in my country and is mainly produced in Shaanxi, Shanxi, Hebei, Inner Mongolia and other regions. According to the 2020 edition of the Chinese Pharmacopoeia, Astragalus complanatus is sweet and warm in nature, and belongs to the liver and kidney meridians. It is widely used to treat kidney deficiency and low back pain, spermatorrhea and premature ejaculation, enuresis and frequent urination, leucorrhea, dizziness, and blurred vision. Studies in recent years have shown that Astragalus complanatus is rich in flavonoid functional active ingredients, including Astragalus complanatus glycoside and Astragalus complanatus glycoside B. Astragalus complanatus glycoside has multiple biological activities such as anti-inflammatory, liver protection, anti-cancer, anti-aging, intervention in pulmonary fibrosis, treatment of vitiligo, and skin whitening. Therefore, it is of great practical value to develop an extraction method for Astragalus complanatus glycoside in Astragalus complanatus.

[0003] At present, the existing methods for extracting complanatus glycosides mainly use ethanol as the extraction solvent, and adopt methods such as heating reflux extraction, ultrasonic extraction, microwave extraction, and percolation extraction. However, organic solvents have many disadvantages, such as low efficiency, environmental pollution, and potential safety hazards. Therefore, it is an urgent problem to develop new green solvents to replace organic solvents to extract complanatus glycosides from complanatus glycosides.

[0004] DES was first proposed in 2001 and is mainly composed of hydrogen bond acceptors (HBA) and hydrogen bond donors (HBD). As a new type of solvent, DES has significant advantages such as easy preparation, low cost, low toxicity, low volatility, and high extraction rate. Although there are many reports on the application of DES in the extraction of natural products, there are no reports on the extraction and preparation of complanatus glycosides.

[0005] In addition, as a type of material commonly used for natural product separation, macroporous adsorption resin has many advantages such as good adsorption selection, recyclability, simple operation, and low cost. The invention patent of Chinese patent application number CN200910057073.3 discloses a method for adsorbing and separating complanatus glycoside from 80% ethanol water extract of Astragalus complanatus by using macroporous adsorption resin. The invention patent of Chinese patent application number CN201010603920.4 discloses a method for adsorbing and separating complanatus glycoside from alkaline water or alkaline dilute alcohol extract of Astragalus complanatus by using macroporous adsorption resin. Therefore, it is feasible to use macroporous adsorption resin to enrich complanatus glycoside after DES extraction. Summary of the invention

[0006] In view of the problems existing in the prior art, the purpose of the present invention is to design and provide a method for extracting complanatus glycoside from Astragalus complanatus, and extracting complanatus glycoside from Astragalus complanatus by using DES and macroporous adsorption resin. The method firstly optimizes the extraction conditions by optimizing the deep eutectic solvent and efficiently obtains the complanatus glycoside extract; then uses the macroporous adsorption resin for adsorption separation to separate and obtain complanatus glycoside from the complanatus glycoside extract. The method gives full play to the characteristics of DES and macroporous adsorption resin, and can effectively obtain complanatus glycoside.

[0007] The present invention is achieved through the following technical solutions:

[0008] A method for extracting complanatus glycoside from Complanatus complanatus seeds comprises the following steps:

[0009] 1) preparing DES: eutectic a hydrogen bond acceptor and a hydrogen bond donor to obtain DES (Deep Eutectic Solvents), and letting the mixture stand, wherein the hydrogen bond acceptor is at least one of choline chloride, L-proline and betaine; the hydrogen bond donor is at least one of levulinic acid, ethylene glycol, fructose, glucose, triethylene glycol, lactic acid, urea, citric acid, glycerol, propylene glycol and sucrose; and the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is (1-2):(1-3);

[0010] 2) DES extraction: the DES obtained in step 1) is mixed with water to prepare an extraction solvent, and the powder of Astragalus complanatus is evenly mixed with the extraction solvent, and subjected to shaking extraction to obtain an extract of Astragalus complanatus glycosides;

[0011] 3) Separation of Compasoside by Macroporous Adsorption Resin: The Compasoside Extract obtained in step 2) is loaded onto a macroporous adsorption resin, eluted with an eluent, and concentrated to obtain crude Compasoside;

[0012] 4) Purification of Compasoside by recrystallization: The crude Compasoside obtained in step 3) is recrystallized with an organic solvent to obtain pure Compasoside.

[0013] Furthermore, the method further comprises 5) DES recovery: collecting the sample effluent from step 3), evaporating the water in the effluent, and reusing it for extracting complanatus glycosides from Complanatus complanatus, thus completing the recovery of the low eutectic solvent.

[0014] Furthermore, in step 1), the hydrogen bond acceptor is L-proline; the hydrogen bond donor is levulinic acid; the molar ratio of L-proline to levulinic acid is 1:(1-3), specifically, the molar ratio of L-proline to levulinic acid is 1:2.

[0015] Furthermore, the eutectic treatment conditions in step 1) are: stirring in a water bath at 60-80° C. for 1-3 hours and standing for 8-15 hours.

[0016] Furthermore, in the step 2), the particle size of the Astragalus complanatus powder is less than 1 mm.

[0017] Furthermore, in the step 2), DES and water are mixed to form an extraction solvent with a water content of 10-50%; the ratio of the extraction solvent to the powder of Astragalus complanatus is 5-30 mL / g; and the extraction conditions are: 30-40° C., 100-500W ultrasonic extraction for 20-60 min.

[0018] Preferably, in the step 2), DES and water are mixed to form an extraction solvent with a water content of 30-40%, and the liquid ratio of the extraction solvent to the Astragalus complanatus powder is 20-25 mL / g.

[0019] Furthermore, in the step 3), the macroporous adsorption resin model is ADS-17, XAD-76, DM301, NKA-9, HPD-1100, D101 or AB-8.

[0020] Furthermore, in the step 3), the adsorption separation conditions of the macroporous adsorption resin are: solid-liquid ratio of 0.33-3 g / mL, initial loading concentration of 20-100 μg / mL, and adsorption temperature of 20-30°C.

[0021] Furthermore, in the step 3), eluting with an eluent means eluting with distilled water, 50v% ethanol, 70v% ethanol, and 90v% ethanol in sequence, collecting the 70v% ethanol eluate, and concentrating it to obtain the crude product of complanatus glycosides.

[0022] Furthermore, in the step 4), the organic solvent refers to an organic solvent composed of dichloromethane and methanol in a volume ratio of (1-3):1.

[0023] The present invention can obtain the complanatus glycoside compound from the complanatus scutellariae simply and efficiently, and the extracted complanatus glycoside yield is high, which is more than 10 times the extraction rate of traditional organic solvents. The extraction method provided by the present invention uses DES, which is a mixed salt solution formed by two or more cheap and safe compounds combined with each other through hydrogen bonding. DES has a large polarity and its physical and chemical properties are close to those of ionic liquids, but it is safer, cheaper, and easier to prepare than ionic liquids, and the extraction procedure is simple, the extraction efficiency is high, and the analysis time is short. Compared with traditional organic solvents, DES has the advantages of low raw material price, good biocompatibility, low volatility, simple operation process, and reusability, which fully reflects the characteristics of green chemistry. Secondly, the macroporous adsorption resin used in the present invention can be reused repeatedly, and the operation process is simple, and it has good prospects for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the HPLC spectrum of the standard product of Astragalus complanatus glycoside;

[0025] Figure 2 This is the HPLC spectrum of DES-extracted Astragalus complanatus glycosides;

[0026] Figure 3 The effects of DES water content (a), liquid-to-solid ratio (b), extraction temperature (c), and extraction time (d) on the extraction rate of Astragalus complanatus glycosides;

[0027] Figure 4 To optimize the effect of interaction on the extraction efficiency of complanatus glycosides from Astragalus complanatus by response surface method;

[0028] Figure 5 is the recovery rate of astragalus glycoside adsorbed by different macroporous adsorption resins;

[0029] Figure 6 To investigate the effects of solid-liquid ratio (a) and initial loading concentration (b) of AB-8 macroporous adsorption resin on the adsorption rate of astragalus glycosides;

[0030] Figure 7 HPLC chart of the glycosides of Astragalus membranaceus obtained by purification by recrystallization after recovery by AB-8 macroporous adsorption resin in Example 3;

[0031] Figure 8 The chemical structure diagram of Compasaena glycoside;

[0032] Fig. 9 Complanatus glycoside 1 H-NMR spectrum;

[0033] Fig.10 Complanatus glycoside 13 C-NMR spectrum;

[0034] Fig.11 These are SEM photos of the untreated Astragalus complanatus powder sample, the Astragalus complanatus powder sample after DES extraction in Example 3, and the Astragalus complanatus powder sample after ethanol extraction in Comparative Example 1. DETAILED DESCRIPTION

[0035] The present invention is further described below with reference to specific embodiments.

[0036] The HPLC conditions used in this example to analyze the complanatus glycosides in the complanatus glabra extract are:

[0037] Chromatographic column: Agilent XDB-C18 column (4.6×250 mm, 5 μm), flow rate: 1 mL / min, column temperature: 25°C, detection wavelength: 266 nm, injection volume: 20 μL, mobile phase: acetonitrile (A)-0.1% phosphoric acid water (B), elution conditions: 0-25 min, 20% A. The HPLC detection spectrum of the standard complanatus glycosides is shown in Figure 1 .

[0038] Example 1: Selection of DES and selection of DES extraction conditions

[0039] (1) Preparation of DES: The hydrogen bond acceptor and the hydrogen bond donor were uniformly mixed in a certain molar ratio. The hydrogen bond acceptor and the hydrogen bond donor and their molar ratio are shown in Table 1. The mixture was placed in a sealed glass bottle and stirred in a water bath at 70°C at 500 rpm for 1 to 3 hours to obtain a pure uniform liquid. The mixture was then allowed to stand at 30°C (room temperature) for 12 hours. A total of 17 types of DES were prepared in this embodiment.

[0040] (2) DES extraction: The solution obtained in (1) was prepared into an extraction solvent A with a water content of 20% (volume ratio), and the extraction solvent A was mixed with the complanatus complanatus powder at a liquid-solid ratio of 8 mL / g. The mixture was extracted at 60°C and 250W ultrasound for 20 min to obtain the complanatus complanatus glycoside extract. The complanatus complanatus glycoside yield was calculated by HPLC (Table 1). As shown in Table 1, the extraction rate of complanatus complanatus glycoside with DES No. 17 (L-proline:levulinic acid = 1:2) was the highest, which was 1.267%. The HPLC detection spectrum is shown in Table 1. Figure 2 Therefore, the present invention selects DES No. 17 as the extraction solvent.

[0041] Table 1 Compositions of different DES and their extraction rates of glycosides from Astragalus complanatus

[0042]

[0043] (a) Selection of DES moisture content

[0044] No. 17 DES was used as the extraction solvent. According to the above extraction conditions, other experiments were the same, and the effects of DES water content of 10%, 20%, 30%, 40%, 50%, and 60% on the extraction efficiency of complanatus glycosides were investigated. Figure 3 As shown in (a), when the water content is 40%, the extraction efficiency reaches the maximum value, and the extraction efficiency decreases when the water content continues to increase. In summary, the present application selects a DES water content of 30-40%.

[0045] (b) Selection of liquid-to-solid ratio

[0046] No. 17 DES was used as the extraction solvent. According to the above extraction conditions, other experiments were the same, and the effects of different liquid-to-solid ratios of 5 mL / g, 10 mL / g, 15 mL / g, 20 mL / g, 25 mL / g, and 30 mL / g on the extraction efficiency of complanatus glycosides were investigated. Figure 3

[0047] As shown in (b), when the liquid-to-solid ratio reaches 25 mL / g, the extraction efficiency reaches the maximum value. If the solid-to-liquid ratio is further increased, the extraction efficiency will decrease. This may be due to the results and physical properties of DES itself. In summary, the liquid-to-solid ratio is selected as 20-25 mL / g in this application.

[0048] (c) Selection of extraction temperature

[0049] No. 17 DES was used as the extraction solvent. According to the above extraction conditions, other experiments were the same, and the effects of different extraction temperatures of 30℃, 40℃, 50℃, 60℃, and 70℃ on the extraction efficiency of the target compound were investigated. Figure 3 As shown in (c), when the extraction temperature increases from 30°C to 40°C, the extraction efficiency of complanatin gradually increases, while when the temperature further increases, the extraction efficiency of complanatin sharply decreases. In summary, the present application selects an extraction temperature of 30-40°C.

[0050] (d) Selection of extraction time

[0051] No. 17 DES was used as the extraction solvent. According to the above extraction conditions, other experiments were the same, and the effects of different extraction times of 10 min, 20 min, 30 min, 40 min, and 50 min on the extraction efficiency of complanatus glycosides were investigated. Figure 3 As shown in (d), when the extraction time is 30 minutes, the extraction efficiency of the target compound is the highest. After further extending the extraction time, the extraction efficiency of the target compound does not change significantly, indicating that the extraction process has reached equilibrium. In summary, the extraction time selected in this application is 20 to 30 minutes.

[0052] (e) Optimization of the optimal extraction conditions by response surface experiment

[0053] According to the central composite experimental design principle of Box-Behnken, the extraction rate of complanatus glycosides was taken as the response value (Y), the extraction temperature was fixed at 30°C, the DES moisture content (A), liquid-to-solid ratio (B), and extraction time (C) were selected as optimization factors, and the response surface analysis method with three factors and three levels was adopted. The experimental factors and level designs are shown in Table 2.

[0054] Table 2 Response surface factor design level table

[0055]

[0056]

[0057] In order to investigate the influence of various factors and their interactions on the extraction efficiency, the present invention uses Design-Expert software to perform multiple regression fitting on the four factors and three levels in the response surface factor design level table to obtain the calculation equation of the total extraction rate:

[0058] R1=1.38-0.1041A+0.0758B-0.0583C+0.0844AB-0.1067AC+0.0313BC-0.7856A 2 -0.1514B2 -

[0059] 0.3152C 2 .

[0060] The variance analysis of the quadratic polynomial model is shown in Table 3. The model P<0.0002 (extremely significant), indicating that the model can be used to predict the response value. The model lack of fit term is not significant, indicating that the regression equation has a good degree of fit in the entire regression space.

[0061] Table 3 Model ANOVA

[0062]

[0063] The response surface diagram obtained based on the regression equation is as follows: Figure 4 As shown, the relationship between the effects and interactions of various factors and the extraction rate is shown. Through analysis, it is concluded that the optimal extraction conditions for complanatine glycosides in Complanatine Fructus are: water content 38%, extraction time 30min, and liquid-to-solid ratio 26mL / g. Under these conditions, the extraction rate of complanatine glycosides was 1.56% (15.60mg / g), which is close to the calculated value of 1.29% (12.88mg / g) of the model, indicating that the optimal extraction conditions obtained by the response surface methodology are accurate and reliable, and have practical application value.

[0064] Example 2: Selection of macroporous adsorption resin and selection of loading conditions

[0065] Macroporous adsorption resin separation: Take 1 mL of the complanatus glycoside extract obtained by using No. 17 DES as the extractant in Example 1, load it on a macroporous adsorption resin, and elute it with distilled water, 50% ethanol, 70% ethanol, and 90% ethanol in sequence, collect the 70% ethanol eluate, and concentrate it to obtain the crude complanatus glycoside.

[0066] (a) Screening of macroporous adsorption resins

[0067] Seven macroporous adsorption resins, ADS-17, XAD-76, DM301, NKA-9, HPD-1100, D101 and AB-8, were selected to conduct adsorption and recovery experiments on the extract of complanatus glycosides. The results showed that AB-8 had the best effect on complanatus glycosides ( Figure 5 a), AB-8 was reused 5 times, and the recovery rate was good ( Figure 5 b).

[0068] (b) Study on the influence of sample loading conditions

[0069] Specifically, according to the above adsorption and elution steps, other conditions were the same, and the effects of the ratio of macroporous adsorption resin to complanatus glycoside extract (1 mL) (solid-liquid ratio of 3, 2, 1, 0.5, 0.33 g / mL) and the loading concentration (300, 200, 100, 50, 25 μg / mL) on the adsorption rate of complanatus glycoside were investigated. The results showed that the solid-liquid ratio was between 1 and 3 g / mL ( Figure 6 a), the loading concentration is 25~100μg / mL ( Figure 6 b) has a better adsorption rate. Therefore, selecting a suitable solid-liquid ratio, adsorption concentration and adsorption temperature can increase the recovery rate of complanatus glycosides.

[0070] Example 3: A method for extracting complanatus glycoside from Complanatus complanatus

[0071] 1) Preparation of DES: L-proline, a hydrogen bond acceptor, and levulinic acid, a hydrogen bond donor, were eutectic at a molar ratio of 1:2. The eutectic conditions were: stirring at 500 rpm and 70° C. in a water bath for 1 hour to obtain DES, and then standing for 14 hours;

[0072] 2) DES extraction: The DES obtained in step 1) was mixed with water to prepare an extraction solvent with a water content of 40%, and the extraction solvent was evenly mixed with 10 g of Complanthus Complanthus powder at a liquid-to-solid ratio of 25 mL / g. The particle size of the Complanthus Complanthus powder was less than 1 mm. The powder was extracted at 40° C. and 250 W ultrasound for 30 min to obtain an extract of Complanthus Complanthus glycosides. The yield of Complanthus Complanthus glycosides in the extract was 1.57% as determined by HPLC.

[0073] 3) Macroporous adsorption resin adsorption separation and recrystallization: Take the complanatus glycoside extract obtained in step 2) and load it on the macroporous adsorption resin. The macroporous adsorption resin model is AB-8. The macroporous adsorption resin adsorption separation conditions are: solid-liquid ratio is 3g / mL, initial loading concentration is 25μg / mL, and distilled water, 50v% ethanol, 70v% ethanol, and 90v% ethanol are used for elution in sequence. The 70v% ethanol eluate is collected and concentrated to obtain the complanatus glycoside crude product. The complanatus glycoside crude product is recrystallized with dichloromethane / methanol (volume ratio 2:1) to obtain 153.28mg of complanatus glycoside with a purity of 89.12% (HPLC chart see Figure 7 ), and its structure was confirmed to be complanatus glycoside by NMR, and its structure is as follows Figure 8 As shown, NMR diagram is shown in Fig. 9 and Fig.10 .

[0074] Instrument materials: The melting point was determined by a micro melting point instrument SGWX-4A. 1 H-NMR and 13 C-NMR spectra were measured by a Bruker AVANCE III HD 400 nuclear magnetic resonance instrument.

[0075] Experimental results: Complanatus glycoside: molecular formula C 28 H 32 O6. Yellow powdery solid, melting point 279-280℃. It has strong fluorescence under UV254nm lamp, and 10% sulfuric acid ethanol color developer turns it yellow. 1 H-NMR (DMSO-d6, 400MHz) δ: 12.56 (1H, s, 5-OH), 8.16 (2H, d, J = 8.8Hz, H-2′, 6′), 7.17 (2H, J = 8.8Hz, H-3′, 5′), 6.78 (1H, d , J=2.4Hz, H-8), 6.40 (1H, d, J=2.0Hz, H-6), 5.50 (1H, d, J=7.2Hz, H-1″), 5.04 (1H, d, J=7.2Hz, H-1), 3.87 (3H, s, 7-OMe). 13 C-NMR (DMSO-d6, 100MHz) δ: 178.2 (C-4), 165.7 (C-7), 161.4 (C-5), 159.8 (C-4′), 156.9 (C-2 ), 156.4(C-9), 134.4(C-3), 131.1(C-2′), 124.1(C-1′), 116.3(C-3′), 105.6(C-10), 101.2 (C-1″), 100.4(C-1), 98.5(C-6), 92.9(C-8), 78.1(C-5), 77.6(C-5″), 77.0(C-3″), 76.9(C- 3), 74.7(C-2″), 73.7(C-2), 70.4(C-4), 70.1(C-4″), 61.3(C-6), 61.1(C-6″), 56.6(7-OMe).

[0076] Example 4: A method for extracting complanatus glycoside from Complanatus complanatus

[0077] 1) collecting the sample effluent in Example 3 (about 5 times the volume of the sample), evaporating the water in the effluent under reduced pressure using a rotary evaporator, and mixing with water to prepare an extraction solvent with a water content of 40%, and repeatedly using the solvent to extract the complanatine from the complanatine;

[0078] 2) extracting the complanatus glycosides in the complanatus complanatus again from the recovered DES with a water content of 40% according to the extraction steps and conditions of the above-mentioned Example 3 to obtain an extract;

[0079] 3) the extract was adsorbed, recovered and recrystallized according to the above Example 3 to obtain recovered DES and complanatus glycosides;

[0080] 4) Repeat the operation 5 times.

[0081] The test results of Example 4 are shown in Table 4. After the DES prepared by the present invention was recovered, when the complanatus glycoside in the complanatus glycoside was extracted for the third time, the extraction efficiency was 80.24% of the original, and it still had a good extraction effect, indicating that the DES adsorbed by the AB-8 macroporous resin has good solvent integrity and can be recycled at least 3 times, thereby saving costs to a greater extent.

[0082] Table 4 Extraction effect of DES reused 5 times

[0083]

[0084] Note: Extraction efficiency (%) = (pure product mass / 136.60 mg) × 100; wherein 136.60 mg is the mass of complanatus glycoside with a purity of 100% obtained in Example 2: purity 89.12% × weight 153.28 mg = 136.60 mg.

[0085] Example 5: A method for extracting complanatus glycoside from Complanatus complanatus

[0086] 1) Preparation of DES: L-proline, a hydrogen bond acceptor, and levulinic acid, a hydrogen bond donor, were eutectic at a molar ratio of 1:2. The eutectic conditions were: stirring at 500 rpm in a 70° C. water bath for 1 hour to obtain DES, and then standing for 16 hours;

[0087] 2) DES extraction: The DES obtained in step 1) was mixed with water to prepare an extraction solvent with a water content of 35%, and then the extraction solvent was mixed evenly with 10 g of Compasaena complanata powder at a liquid-to-solid ratio of 20 mL / g, wherein the particle size of the Compasaena complanata powder was less than 1 mm. The powder was extracted at 30° C. and 250 W ultrasound for 25 min to obtain an extract of Compasaena complanata glycosides. The yield of Compasaena complanata glycosides in the extract was determined by HPLC to be 1.49%.

[0088] 3) Macroporous adsorption resin adsorption separation and recrystallization: Take the complanatus glycoside extract obtained in step 2) and load it on a macroporous adsorption resin. The macroporous adsorption resin model is D-101. The macroporous adsorption resin adsorption separation conditions are: solid-liquid ratio is 2g / mL, initial loading concentration is 100μg / mL, and distilled water, 50% ethanol, 70% ethanol, and 90% ethanol are used for elution in sequence. The 70% ethanol eluate is collected and concentrated to obtain a crude complanatus glycoside. The crude complanatus glycoside is recrystallized with dichloromethane / methanol (volume ratio 2:1) to obtain 147.87 mg of complanatus glycoside with a purity of 88.95%.

[0089] Comparative Example 1: A method for extracting complanatus glycoside from complanatus scutellariae using ultrasound-assisted ethanol

[0090] 60% ethanol extraction: 60v% ethanol and 10g of Compasaenae Astragali powder were mixed evenly at a liquid-to-solid ratio of 20mL / g. The particle size of the Compasaenae Astragali powder was less than 1mm. The powder was extracted at 40°C and 250W ultrasound for 30min to obtain an extract of Compasaenae glycoside. The yield of Compasaenae glycoside in the extract was 0.147% as detected by HPLC.

[0091] The results of the complanatine content in the extracts of Example 3, Example 5 and Comparative Example 1 are shown in Table 5. The results show that the extraction rate of complanatine using DES as the extraction solvent is significantly higher than the extraction rate using ethanol as the solvent.

[0092] Table 5 Comparison of the content of glycosides in different extraction methods

[0093]

[0094] The untreated powder sample of Astragalus complanatus, the powder sample of Astragalus complanatus extracted with DES in Example 3, and the powder sample of Astragalus complanatus extracted with ethanol in Comparative Example 1 were analyzed by scanning electron microscope (instrument model: FEI NovaNanoSEM450). The results are as follows: Fig.11 a, 11b and 11c. For the dried untreated samples, the sample surface was relatively intact. After extraction with DES or ethanol, the sample surface showed visible pores and cracks, and the surface morphology of the sample was destroyed. This damage was more obvious with DES, indicating that DES can more effectively destroy cells, thereby releasing more compounds in the material.

[0095] The above description is only a preferred embodiment of the present invention, but the present invention is not limited to the scope of implementation shown. Any changes made in accordance with the concept of the present invention, or modifications to equivalent embodiments with equivalent changes, which still do not exceed the spirit covered by the specification, should be within the protection scope of the present invention.

Claims

1. A method for extracting complanatus glycoside from Complanatus complanatus, characterized in that: The steps include: 1) Preparation of DES: co-melting a hydrogen bond acceptor and a hydrogen bond donor to obtain DES, and letting it stand; the hydrogen bond acceptor is at least one of choline chloride, L-proline and betaine; the hydrogen bond donor is at least one of levulinic acid, ethylene glycol, fructose, glucose, triethylene glycol, lactic acid, urea, citric acid, glycerol, propylene glycol and sucrose; the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is (1-2): (1-3); 2) DES extraction: the DES obtained in step 1) is mixed with water to form an extraction solvent, and then the Compasaenae Astragali powder and the extraction solvent are evenly mixed, and ultrasonic extraction is performed to obtain an extract of Compasaenae Astragali glycosides; 3) Separation of Compasoside by Macroporous Adsorption Resin: The Compasoside Extract obtained in step 2) is loaded onto a macroporous adsorption resin, eluted with an eluent, and concentrated to obtain crude Compasoside; 4) Purification of Compasoside by recrystallization: The crude Compasoside obtained in step 3) is recrystallized with an organic solvent to obtain pure Compasoside.

2. The method for extracting complanatus glycoside from Complanatus complanatus according to claim 1, characterized in that: The method further comprises step 5) DES recovery: collecting the sample effluent from step 3), evaporating the water in the effluent, and reusing it for extracting complanatus glycosides from complanatus scutellariae, thus completing the recovery of the low eutectic solvent.

3. The method for extracting complanatus glycoside from Fructus Astragali according to claim 1, characterized in that: The eutectic treatment conditions in step 1) are: stirring in a water bath at 60-80° C. for 1-3 hours.

4. The method for extracting complanatus glycoside from Complanatus complanatus according to claim 1, characterized in that: In the step 2), the particle size of the Astragalus complanatus powder is less than 1 mm.

5. The method for extracting complanatus glycoside from Complanatus complanatus according to claim 1, characterized in that: In the step 2), DES and water are mixed to form an extraction solvent with a water content of 10-50%; the ratio of the extraction solvent to the powder of Astragalus complanatus is 5-30 mL / g; and the extraction conditions are: 30-40° C., extraction under 100-500W ultrasound for 20-60 min.

6. The method for extracting complanatus glycoside from Complanatus complanatus according to claim 1, characterized in that: In the step 3), the model of the macroporous adsorption resin is ADS-17, XAD-76, DM301, NKA-9, HPD-1100, D101 or AB-8, and the adsorption separation conditions of the macroporous adsorption resin are: solid-liquid ratio of 0.33-3 g / mL, and initial loading concentration of 20-100 μg / mL.

7. The method for extracting complanatus glycoside from Complanatus complanatus according to claim 1, characterized in that: In the step 3), eluting with an eluent means eluting with distilled water, 50% ethanol, 70% ethanol, and 90% ethanol in sequence, collecting the 70% ethanol eluate, and concentrating it to obtain the crude product of complanatus glycoside.

8. The method for extracting complanatus glycoside from Complanatus complanatus according to claim 1, characterized in that: In the step 4), the organic solvent refers to an organic solvent composed of dichloromethane and methanol in a volume ratio of (1-3):

1.

9. The method for extracting complanatus glycoside from Complanatus complanatus according to claim 1, characterized in that: In the step 1), the hydrogen bond acceptor is L-proline; the hydrogen bond donor is levulinic acid; and the molar ratio of L-proline to levulinic acid is 1:(1-3).

10. The method for extracting complanatus glycoside from Complanatus complanatus according to claim 5, characterized in that: DES and water were mixed into an extraction solvent with a water content of 30-40%; the liquid ratio of the extraction solvent to the Astragalus complanatus powder was 20-30 mL / g; the extraction conditions were: 30-40°C, 100-500W ultrasonic extraction for 20-60 min.

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