A method for extracting five medicinal ingredients from Pueraria root based on deep eutectic solvent

Abstract

The present invention provides a method for extracting five kinds of medicinal ingredients in Pueraria root based on a low eutectic solvent. The preparation of the low eutectic solvent adopts a one-step preparation method, and a hydrogen bond donor and an acceptor according to a certain molar ratio are mixed until a stable liquid is formed; then the low eutectic solvent is added to the pre-treated Pueraria root, and after preparing a sample solution, HPLC detection is performed. Among them, the five medicinal ingredients in Pueraria root are 3-hydroxypuerarin, puerarin, 3-methoxypuerarin, daidzin, and daidzein; ultrasound is used to assist in the preparation of the sample solution; the hydrogen bond donor of the low eutectic solvent is urea, fructose, or glucose, and the acceptor is choline chloride or betaine. The preparation of the low eutectic solvent of the present invention is simple and economical, does not require auxiliary technology, does not require purification, and adopts a one-step preparation method.

Description

Technical Field

[0001] The invention relates to the technical field of natural organic compound extraction and separation, and in particular to a method for extracting five medicinal ingredients from kudzu root based on a low eutectic solvent. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Pueraria root is also known as wild kudzu. In some Western countries, kudzu root extracts are used in the medical and health care industry. Pueraria root total flavonoids extract can increase the content of female estrogen and treat dysmenorrhea. Pueraria root can also treat symptoms such as headache, fever, heartache, etc. Pueraria root contains essential elements needed by humans such as calcium, iron, zinc, etc. The main nutrients in Pueraria root include 3-hydroxypuerarin, puerarin, daidzein, and daidzein. Pueraria root powder, which is composed of five Chinese medicinal materials such as Guanzhong, dried kudzu flower, Pueraria root, licorice, and Amomum villosum, comes from Volume 12 of "Rumen Shiqin" and is used to treat excessive drinking and internal alcohol poisoning.

[0004] Isoflavone compounds are the main biologically active ingredients in Pueraria lobata, among which puerarin has the highest content. At present, there are two types of research on the extraction of puerarin from Pueraria lobata. One is the research on the extraction of flavonoids from Pueraria lobata. The other is the research on the optimization of the extraction method of total flavonoids from Pueraria lobata. However, in many studies, few use deep eutectic solvents for the extraction of active ingredients. Deep eutectic solvents have stable chemical properties, high thermal stability, and large heat capacity, and have received extensive attention from scientists in various fields of chemistry.

[0005] At present, industrial pollution is becoming increasingly serious. It is increasingly urgent to study the use of new green solvents to extract the active ingredients in natural compounds and reduce environmental pollution. Summary of the invention

[0006] In order to solve the deficiencies of the prior art, the present invention provides a method for extracting five medicinal ingredients from Pueraria root based on a deep eutectic solvent. According to the influence of the deep eutectic solvent on the extraction efficiency, daidzein, daidzin, 3-methoxypuerarin, puerarin, and 3-hydroxypuerarin in Pueraria root are extracted, the total flavonoid content is calculated, and the most suitable extraction conditions are obtained.

[0007] In order to achieve the above object, the technical solution of the present invention is:

[0008] The first aspect of the present invention provides a method for extracting five medicinal ingredients from Pueraria root based on a low eutectic solvent, characterized in that the preparation of the low eutectic solvent adopts a one-step preparation method, mixing a hydrogen bond donor and an acceptor according to a certain molar ratio until a stable liquid is formed; taking a certain amount of pre-treated Pueraria root and placing it in a colorimetric tube, then adding the low eutectic solvent, and then placing the colorimetric tube in a digitally controlled ultrasonic cleaner for ultrasonication, shaking and standing, and then centrifuging, taking the supernatant to prepare a sample solution, and performing HPLC detection;

[0009] The hydrogen bond donor is urea, citric acid, fructose or glucose, the acceptor is choline chloride or betaine, and the mass ratio of the kudzu root to the low eutectic solvent is 1:1

[0010] The second aspect of the present invention provides a low eutectic solvent used in a method for extracting five medicinal ingredients from Pueraria root based on the above-mentioned low eutectic solvent.

[0011] The beneficial effects of the present invention are:

[0012] (1) The preparation of the low eutectic solvent of the present invention is simple and economical, and does not require auxiliary technology or purification. It adopts a one-step preparation method to mix two or more compounds until a stable liquid is formed. The low eutectic solvent is relatively stable to water and air, and is generally prepared at room temperature and pressure, and basically does not require a special environment such as a vacuum or an inert atmosphere.

[0013] (2) According to the influence of the low eutectic solvent on the extraction efficiency, the present invention extracts daidzein, daidzein, 3-methoxypuerarin, puerarin, and 3-hydroxypuerarin from Pueraria root, calculates the total flavonoid content, and obtains the most suitable extraction conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0015] Figure 1 This is a standard curve diagram of 3-hydroxypuerarin in Example 1 of the present invention.

[0016] Figure 2 This is the standard curve of puerarin in Example 1 of the present invention.

[0017] Figure 3 This is a standard curve diagram of 3-methoxypuerarin in Example 1 of the present invention.

[0018] Figure 4 This is a standard curve diagram of daidzein in Example 1 of the present invention.

[0019] Figure 5This is a standard curve diagram of daidzein in Example 1 of the present invention.

[0020] Figure 6 The figure is a high performance liquid chromatogram of the extraction of five active ingredients from Pueraria root in Example 2 of the present invention.

[0021] Figure 7 The figure shows the effect of temperature on the extraction rate of puerarin in Example 3 of the present invention.

[0022] Figure 8 The figure shows the effect of temperature on the extraction rate of 3-hydroxypuerarin in Example 3 of the present invention.

[0023] Fig. 9 The figure shows the effect of temperature on the extraction rate of 3-methoxypuerarin in Example 3 of the present invention.

[0024] Fig.10 This is the effect of temperature on the extraction rate of daidzein in Example 3 of the present invention.

[0025] Fig.11 This is the effect of temperature on the extraction rate of daidzein in Example 3 of the present invention.

[0026] Fig.12 This is the effect of time on the extraction rate of 3-hydroxypuerarin in Example 4 of the present invention.

[0027] Fig.13 This is the effect of time on the extraction rate of puerarin in Example 4 of the present invention.

[0028] Fig.14 This is the effect of time on the extraction rate of 3-methoxypuerarin in Example 4 of the present invention.

[0029] Fig.15 This is the effect of time on the extraction rate of daidzein in Example 4 of the present invention.

[0030] Fig.16 This is the effect of time on the extraction rate of daidzein in Example 4 of the present invention.

[0031] Fig.17 The figure shows the effect of the amount of water on the extraction rate of 3-hydroxypuerarin in Example 5 of the present invention.

[0032] Fig.18 This is the effect of the amount of water on the extraction rate of puerarin in Example 5 of the present invention.

[0033] Fig.19 The figure shows the effect of the amount of water on the extraction rate of 3-methoxypuerarin in Example 5 of the present invention.

[0034] Fig. 20 This is the effect of the amount of water on the extraction rate of daidzein in Example 5 of the present invention.

[0035] Fig.21 This is the effect of the amount of water on the extraction rate of daidzein in Example 5 of the present invention. DETAILED DESCRIPTION

[0036] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0037] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0038] At present, industrial pollution is becoming increasingly serious. It is increasingly urgent to study new green solvents and reduce environmental pollution. The main purpose of this topic is to study the process of extracting 5 active ingredients from Pueraria root with low eutectic solvents, deeply study the effect of low eutectic solvents on extraction efficiency, extract daidzein, daidzin, 3-methoxypuerarin, puerarin, 3-hydroxypuerarin from Pueraria root, calculate the total flavonoids content, and obtain the most suitable extraction conditions. Provide direction for efficient and green extraction of other Chinese medicinal materials.

[0039] The present invention takes Pueraria root as the research object. Pueraria root contains active ingredients such as 3-hydroxypuerarin, puerarin, daidzein, daidzein, 3-methoxypuerarin and the like. Ultrasound-assisted low eutectic solvent is used to extract the active ingredients, so as to achieve a certain drug therapeutic effect and improve its practical application in industry.

[0040] The invention provides a method for extracting five medicinal ingredients from kudzu root based on a low eutectic solvent. The preparation of the low eutectic solvent adopts a one-step preparation method, wherein a hydrogen bond donor and an acceptor in a certain molar ratio are mixed until a stable liquid is formed; a certain amount of pre-treated kudzu root is placed in a colorimetric tube, and the low eutectic solvent is added, and then the colorimetric tube is placed in a digitally controlled ultrasonic cleaner for ultrasonication, and after being shaken and allowed to stand, centrifuged, and the supernatant is taken to prepare a sample solution, and HPLC detection is performed;

[0041] The hydrogen bond donor is urea, citric acid, fructose or glucose, the acceptor is choline chloride or betaine, and the mass ratio of the kudzu root to the low eutectic solvent is 1:1.

[0042] In some typical embodiments of the present invention, the one-step preparation method is specifically as follows: pouring the hydrogen bond donor and the acceptor into a round-bottom flask according to a certain molar ratio, adding a magnet, placing the round-bottom flask in a heat-collecting constant temperature heating magnetic stirrer, setting the temperature to react for 2-3 hours, and generating a uniform and stable low eutectic solvent;

[0043] Preferably, the reaction time is 2 hours.

[0044] In some typical embodiments of the present invention, the temperature is set as follows: the choline chloride type deep eutectic solvent is set to 80° C., and the betaine type deep eutectic solvent is set to 100° C.;

[0045] The hydrogen bond donors of the choline chloride type deep eutectic solvent are urea, citric acid, glucose and fructose, respectively, and the acceptors are all choline chloride; the hydrogen bond donors of the betaine type deep eutectic solvent are formic acid and acetic acid, respectively, and the acceptors are all betaine.

[0046] In some typical embodiments of the present invention, the hydrogen bond donor is choline chloride and the acceptor is fructose;

[0047] Furthermore, the certain molar ratio is 1:5-3:1; preferably 1:2-2:1.

[0048] In some typical embodiments of the present invention, the pre-treatment steps of the Pueraria root are:

[0049] Put wild kudzu root into a grinder for crushing, and then sieve with sieves of different specifications to obtain samples of different particle sizes, 50-70 mesh, preferably 60-70 mesh, and store the samples in a sample bag in a dry and dark environment;

[0050] Furthermore, the five medicinal ingredients in the Pueraria root are 3-hydroxypuerarin, puerarin, 3-methoxypuerarin, daidzin and daidzein.

[0051] In some typical embodiments of the present invention, the steps of preparing the sample solution are:

[0052] Accurately weigh 99.5 g to 100.5 g of Pueraria root sample into a 1.0 L glass container, add a low eutectic solvent, then add deionized water, put it into a CNC ultrasonic cleaner for ultrasonication, shake it well and let it stand, then centrifuge it, take 1.0 mL of the supernatant, add methanol, mix well, transfer 1.0 mL, and filter through an organic membrane to obtain a sample solution.

[0053] Further, the ultrasonic temperature is 30-70°C, preferably 60°C;

[0054] Furthermore, the ultrasonic time is 5-30 min, preferably 30 min;

[0055] Furthermore, the amount of deionized water added is 1-5 mL, preferably 400 mL;

[0056] Further, the methanol is 3.9 mL of 60% methanol;

[0057] Furthermore, the pore size of the organic membrane is 0.45 μm.

[0058] In some typical embodiments of the present invention, the conditions of the HPLC analysis include: using a GL Sciences InertSustain C18 column with a particle size of 5 μm and an inner diameter of 250 mm×4.6 mm.

[0059] In some typical embodiments of the present invention, the HPLC analysis conditions include: using acetonitrile as the B pump and 0.1% formic acid A pump as the mobile phase;

[0060] The flow rate was 1.0 mL / min;

[0061] The injection volume was 20.0 μL;

[0062] The detection wavelength is λ=260 nm.

[0063] In some typical embodiments of the present invention, the HPLC analysis conditions further include gradient elution:

[0064] 0min: 85% A, 15% B;

[0065] 16min: 70% A, 30% B;

[0066] 30min: 100% B, 0% A;

[0067] 31min: 85% B, 15% A;

[0068] 40min: 85% A, 15% B.

[0069] In another typical embodiment of the present invention, a low eutectic solvent used in a method for extracting five medicinal ingredients from Pueraria root based on the above-mentioned low eutectic solvent is provided.

[0070] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0071] Materials and reagents used in the present invention:

[0072] Pueraria root: Jinan Zhonglu Hospital;

[0073] Methanol: Beijing Bailingwei Technology Co., Ltd.;

[0074] Acetonitrile: Thermo Fisher Scientific (China) Co., Ltd.;

[0075] Formic acid: Tianjin Guangfu Fine Chemical Research Institute;

[0076] Deionized water: Shandong Analysis and Testing Center

[0077] Puerarin standard product: Shanghai Yuanye Biotechnology Co., Ltd.;

[0078] 3-Methoxypuerarin standard: Shanghai Yuanye Biotechnology Co., Ltd.;

[0079] Soybean glycoside standard: Shanghai Yuanye Biotechnology Co., Ltd.;

[0080] Daidzein standard product: Shanghai Yuanye Biotechnology Co., Ltd.;

[0081] 3-Hydroxypuerarin standard: Shanghai Yuanye Biotechnology Co., Ltd.

[0082] The experimental instruments used in the present invention are:

[0083] BPG-9156A precision blast drying oven: Shanghai Jinghong Experimental Equipment Co., Ltd.;

[0084] XS205 electronic balance: METTLER TOLEDO;

[0085] Thermal collector constant temperature heating magnetic stirrer: Gongyi Yingyu High-tech Instrument Factory;

[0086] SPD-20A liquid chromatograph (ultraviolet): Agilent 1260;

[0087] KQ-600KDE CNC ultrasonic cleaning instrument: Kunshan Ultrasonic Instrument Co., Ltd.

[0088] Electric constant temperature water bath: Beijing Yongguangming Medical Instrument Co., Ltd.

[0089] Example 1

[0090] (1) Preparation of deep eutectic solvent

[0091] A hydrogen bond donor such as urea, fructose, glucose and the like and an acceptor such as choline chloride and betaine in a certain molar ratio are poured into a round-bottom flask, a magnet is added, the round-bottom flask is placed in a heat-collecting constant temperature heating magnetic stirrer, the temperature is set (the choline chloride type is set to 80°C, the betaine type is set to 100°C), and the reaction is carried out for about 2 hours to generate a uniform and stable low eutectic solvent.

[0092] (2) Pretreatment of Pueraria root

[0093] The wild kudzu root is put into a grinder for grinding, and then sieved with sieves of different specifications to obtain samples of different particle sizes, 50-70 mesh, preferably 60-70 mesh. In this embodiment, the samples are stored in the mineral sample bags of Shandong Provincial Analysis and Testing Center in a dry and dark environment.

[0094] (3) Preparation of sample solution

[0095] Accurately weigh 100.0 g (± 0.5 g) of Pueraria root sample into a 1.0 L glass container, add a low eutectic solvent, then add 300 mL of deionized water, put it into a CNC ultrasonic cleaner and ultrasonicate it at 30 degrees Celsius for 10 min. After shaking and standing, centrifuge it, take 1.0 mL of the upper clear liquid, add 3.9 mL of 60% methanol, mix well, transfer 1.0 mL, filter it through a 0.45 μm organic membrane to obtain a sample solution, and perform HPLC detection.

[0096] (4) Chromatographic conditions

[0097] HPLC analysis was performed on the Pueraria root sample extract obtained with the deep eutectic solvent and the deep eutectic solvent, and the analysis conditions were as follows: GL Sciences InertSustain C18 column (5 μm, 250 mm×4.6 mm) was used; acetonitrile was used as the B pump and 0.1% formic acid was used as the A pump as the mobile phase

[0098] Gradient elution: 0 min: 85% A, 15% B;

[0099] 16min: 70% A, 30% B;

[0100] 30min: 100% B, 0% A;

[0101] 31min: 85% B, 15% A;

[0102] 40min: 85% A, 15% B;

[0103] The detection wavelength is λ = 260 nm;

[0104] Flow rate: 1.0 mL / min;

[0105] The injection volume was 20.0 μL.

[0106] The retention time, peak area, separation effect were compared and their reproducibility was investigated.

[0107] (5) Drawing of standard curves for five active ingredients in Pueraria root

[0108] Weigh 1.32 mg of 3-hydroxypuerarin into a 10 mL brown volumetric flask and dilute to volume with methanol, M = 0.132 mg / mL;

[0109] Weigh 2.02 mg of puerarin into a 10 mL brown volumetric flask and dilute to volume with methanol, M = 0.202 mg / mL.

[0110] Weigh 2.14 mg of 3-methoxypuerarin into a 10 mL brown volumetric flask and dilute to volume with methanol, M = 0.214 mg / mL.

[0111] Weigh 1.28 mg of daidzein into a 10 mL brown volumetric flask and dilute to volume with methanol, M = 0.128 mg / mL.

[0112] Weigh 1.24 mg of daidzein into a 10 mL brown volumetric flask and dilute to volume with methanol, M = 0.124 mg / mL.

[0113] Take 0.5 mL of 3-hydroxypuerarin with M=0.132 mg / mL and dilute it to 10 mL with 60% methanol to obtain M=6.6 ug / mL.

[0114] Take 2.5 mL of puerarin (M = 0.202 mg / mL) and dilute it to 10 mL with 60% methanol to obtain M = 5.5 ug / mL.

[0115] Take 1 mL of 3-methoxypuerarin (M = 0.214 mg / mL) and dilute it to 10 mL with 60% methanol to obtain M = 21.4 ug / mL.

[0116] Take 0.5 mL of soybean glycoside (M = 0.128 mg / mL) and dilute it to 10 mL with 60% methanol to obtain M = 6.4 ug / mL.

[0117] Take 0.5 mL of daidzein (M = 0.124 mg / mL) and dilute to 10 mL with 60% methanol to obtain M = 6.2 ug / mL. Take 0.2 mL of diluted 3-hydroxypuerarin, puerarin, 3-methoxypuerarin, daidzein and 40 μL of diluted daidzein and 160 μL of 60% methanol.

[0118] Then 3-hydroxy puerarin, puerarin, 3-methoxy puerarin, daidzin and daidzein were tested by HPLC, and the standard curve was drawn with the concentration of the standard as the horizontal axis and the chromatographic peak area as the vertical axis, and the curve equations of 3-hydroxy puerarin, puerarin, 3-methoxy puerarin, daidzin and daidzein were obtained, respectively, as shown in Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 shown.

[0119] Example 2

[0120] Effects of deep eutectic solvents on the extraction efficiency of five active components from Pueraria root

[0121] (1) Accurately weigh 100.0 g (±0.5 g) of Pueraria root sample and put it into a 1.0 L glass container. When the extraction time is 30 min and the particle size of the medicinal material is greater than 70 mesh, different deep eutectic solvents are added to extract the solvents. When the amount of water added is 300 mL, the extraction is performed by HPLC. Other experimental conditions are the same as those in Example 1.

[0122] Among them, the hydrogen bond donor of DES-1 is choline chloride and the acceptor is urea, with a molar ratio of 1:2;

[0123] The hydrogen bond donor of DES-2 is choline chloride, and the acceptor is citric acid, with a molar ratio of 1:2;

[0124] The hydrogen bond donor of DES-3 is choline chloride and the acceptor is glucose, with a molar ratio of 1:2;

[0125] The hydrogen bond donor of DES-4 is choline chloride and the acceptor is fructose, with a molar ratio of 1:2;

[0126] The hydrogen bond donor of DES-5 is betaine and the acceptor is formic acid, with a molar ratio of 1:2;

[0127] The hydrogen bond donor of DES-6 is betaine and the acceptor is acetic acid, with a molar ratio of 1:2.

[0128] After HPLC detection, the chromatogram is as follows Figure 6 The results are shown in Table 1. It can be seen from the table that DES-4 has the best extraction effect.

[0129] Table 1 Extraction efficiency of different deep eutectic solvents

[0130]

[0131]

[0132] Example 3

[0133] Effect of temperature on the extraction efficiency of five flavonoids from Pueraria root

[0134] Accurately weigh 100.0 g (± 0.5 g) of Pueraria root sample, put it in a 1.0 L glass container, add 100.0 g DES-4, add 3 mL of water, and select extraction temperatures of 30°C, 40°C, 60°C, and 70°C for extraction. Other experimental conditions are the same as those in Example 1. The results of HPLC detection are as follows: Figure 7 , Figure 8 , Fig. 9 , Fig.10 and Fig.11 shown.

[0135] It can be seen from HPLC that when 100.0g of low eutectic solvent is added, as the extraction temperature gradually increases, 3-hydroxypuerarin decreases and then increases with the increase of temperature. The extraction rate of puerarin increases first and then decreases, and the extraction rate is the largest at 60℃, that is, the extraction amount of puerarin increases first and then decreases with the increase of extraction temperature, and the extraction amount is the highest at 60℃, and the extraction amount gradually decreases after 60℃. As the temperature increases, some substances decompose and transform into other substances, and the gradual increase in temperature causes partial decomposition of the effective ingredients; the extraction rate of soybean glycoside increases first and then decreases with the increase of extraction temperature, that is, the extraction amount of soybean glycoside increases first and then decreases with the increase of extraction temperature, but the extraction amount is the highest at 60℃, because when the extraction temperature exceeds 60℃, soybean glycoside decomposes into other substances. Therefore, considering the extraction content of 3-hydroxypuerarin, soybean glycoside, soybean aglycone, puerarin, and 3-methoxypuerarin, the extraction content is the highest at 60℃. 60℃ is selected as the optimal extraction temperature.

[0136] Example 4

[0137] Effect of time on the extraction efficiency of five flavonoids from Pueraria root

[0138] Accurately weigh 100.0 g (± 0.5 g) of Pueraria root sample in a colorimetric tube, add 100.0 g of deep eutectic solvent, add 300 mL of water, and select extraction time of 5, 15, 20, and 30 min respectively under the conditions of extraction temperature 30°C, medicinal material particle size greater than 70 mesh, and extraction. Other experimental conditions are the same as those in Example 1. The results are shown in Fig.12 , Fig.13 , Fig.14 , Fig.15 and Fig.16 shown.

[0139] With the extension of extraction time, the contents of 3-hydroxypuerarin, puerarin, 3-methoxypuerarin and daidzein continued to increase, that is, their extraction efficiency was enhanced. With the extension of time, the extraction content of daidzein gradually decreased, and the extraction rate decreased. Taking all factors into consideration, the optimal extraction time was 30 minutes.

[0140] Example 5

[0141] Effect of water volume on the extraction efficiency of five flavonoids from Pueraria root

[0142] Accurately weigh 100.0 g (± 0.5 g) of Pueraria root sample into a colorimetric tube, extract at 30°C for 10 min, add 100.0 g of deep eutectic solvent, add water in the amount of 1.0 mL, 3.0 mL, 4.0 mL, and 5.0 mL respectively under the condition that the particle size of the medicinal material is greater than 70 mesh, and extract by HPLC. Other experimental conditions are the same as those in Example 1. The results are as follows Fig.17 , Fig.18 , Fig.19 , Fig. 20 and Fig.21 As shown. It can be seen from the figure that with the increase of water addition, the extraction rate of daidzein and puerarin increases first and then decreases. When 400mL of water is added, the extraction content of daidzein and puerarin is the highest and the extraction rate is the best. The reason why the extraction volume gradually decreases after exceeding 400mL is that daidzein and puerarin are fat-soluble substances. The increase in the amount of water added leads to a decrease in the amount of 60% methanol used when the extract is fixed to volume, and the fat-soluble effective ingredients are not completely dissolved; the extraction rate of 3-hydroxypuerarin shows an increasing trend with the increase of water addition, that is, the extraction amount of 3-hydroxypuerarin also increases with the increase of water addition. Therefore, after comprehensive consideration, the water addition amount of 400mL is selected.

[0143] Example 6

[0144] Determination of antioxidant activity of Pueraria extract

[0145] This experiment uses the DPPH free radical scavenging method to determine the antioxidant capacity of Pueraria root. The specific steps are as follows:

[0146] Weigh 0.0132g of 2,2-diphenyl-1-1-cobalaminyl free radical and dilute it to a 250mL volumetric flask with anhydrous ethanol, store it in a refrigerator for later use, take 40μL of Pueraria extract in a 4mL colorimetric tube, add 4mL of DPPH, use a constant temperature water bath at 37 degrees Celsius for 20 minutes, and check its absorbance at 510nm with a UV spectrophotometer. Use 4mL of DPPH and 40μL of deionized water as a blank.

[0147] Inhibition rate (%) = [1-(A X -A0') / A0]*100

[0148] Table 2 Antioxidant activity of kudzu root extract

[0149]

[0150] Among them, the components of DES-1, DES-2, DES-3, DES-4, DES-5 and DES-6 and the molar ratio of hydrogen bond donors to acceptors are the same as those in Example 2.

[0151] According to the experimental data of Examples 2-5, when using a low eutectic solvent to extract the five active ingredients of Pueraria lobata, the optimal extraction conditions are time: 30 min, extraction temperature 60 degrees, and medicinal material ratio 1:1; for ionic liquids, the optimal extraction conditions are time: 30 min, extraction temperature 40 degrees, and water addition amount 400 mL.

[0152] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for extracting five medicinal ingredients from Pueraria root based on a deep eutectic solvent, characterized in that: The preparation of the low eutectic solvent adopts a one-step preparation method, mixing a hydrogen bond donor and an acceptor according to a certain molar ratio until a stable liquid is formed; taking a certain amount of pre-treated Pueraria root and placing it in a colorimetric tube, then adding the low eutectic solvent, and then placing the colorimetric tube in a digitally controlled ultrasonic cleaner for ultrasonication, shaking and standing, and then centrifuging, taking the supernatant to prepare a sample solution, and performing HPLC detection; The mass ratio of the kudzu root to the deep eutectic solvent is 1:1; The acceptor of the choline chloride type deep eutectic solvent is choline chloride; the hydrogen bond donor is urea, citric acid, fructose or glucose; the hydrogen bond donor of the betaine type deep eutectic solvent is formic acid and acetic acid, and the acceptor is betaine; The five medicinal ingredients in the pueraria root are 3-hydroxypuerarin, puerarin, 3-methoxypuerarin, daidzin and daidzein; The specific method of the one-step preparation method is as follows: pouring the hydrogen bond donor and the acceptor into a round-bottom flask at a molar ratio of 1:5-3:1, adding a magnet, placing the round-bottom flask in a heat-collecting constant temperature heating magnetic stirrer, setting the temperature to react for 2-3 hours, and generating a uniform and stable low eutectic solvent; The temperature is set as follows: the choline chloride type low eutectic solvent is set at 80°C, and the betaine type low eutectic solvent is set at 100°C.

2. The method for extracting five medicinal ingredients from Pueraria root based on a deep eutectic solvent according to claim 1, characterized in that: The reaction time of the one-step preparation method is 2 hours.

3. The method for extracting five medicinal ingredients from Pueraria root based on a deep eutectic solvent according to claim 1, characterized in that: The hydrogen bond donor is choline chloride, and the acceptor is fructose.

4. The method for extracting five medicinal ingredients from Pueraria root based on a deep eutectic solvent according to claim 1, characterized in that: The certain molar ratio is 1:2-2:

1.

5. The method for extracting five medicinal ingredients from Pueraria root based on a deep eutectic solvent according to claim 1, characterized in that: The pre-treatment steps of the Pueraria root are: Put the wild kudzu root into a grinder for crushing, and then sieve it with sieves of different specifications to obtain samples of different particle sizes, 50-70 mesh, and store the samples in mineral sample bags in a dry and dark environment.

6. The method for extracting five medicinal ingredients from Pueraria root based on a deep eutectic solvent according to claim 5, characterized in that: The samples with different particle sizes are 60-70 mesh.

7. The method for extracting five medicinal ingredients from Pueraria root based on a deep eutectic solvent according to claim 1, characterized in that: The steps for preparing the sample solution are: Accurately weigh 99.5 g to 100.5 g of Pueraria root sample into a 1.0 L glass container, add a low eutectic solvent, then add deionized water, put it into a CNC ultrasonic cleaner for ultrasonication, shake it well and let it stand, then centrifuge it, take 1.0 mL of the supernatant and add methanol, mix well, transfer 1.0 mL, and filter it through an organic membrane to obtain a sample solution; The ultrasonic temperature is 30-70°C; The ultrasonic time is 5-30min; The amount of deionized water added is 100-500 mL; The methanol is 3.9 mL of 60% methanol; The pore size of the organic membrane is 0.45 μm.

8. The method for extracting five medicinal ingredients from Pueraria root based on a deep eutectic solvent according to claim 7, characterized in that: The ultrasonic temperature is 60°C.

9. The method for extracting five medicinal ingredients from Pueraria root based on a deep eutectic solvent according to claim 7, characterized in that: The ultrasonic time is 30 min.

10. The method for extracting five medicinal ingredients from Pueraria root based on a deep eutectic solvent according to claim 7, characterized in that: The amount of deionized water added is 400 mL.

11. The method for extracting five medicinal ingredients from Pueraria root based on a deep eutectic solvent according to claim 1, characterized in that: The conditions of the HPLC analysis include: using a GL Sciences InertSustain C18 column with a particle size of 5 μm and an inner diameter of 250 mm×4.6 mm.

12. The method for extracting five medicinal ingredients from Pueraria root based on a deep eutectic solvent according to claim 11, characterized in that: The HPLC analysis conditions include: using acetonitrile as the B pump and 0.1% formic acid A pump as the mobile phase; The flow rate was 1.0 mL / min; The injection volume was 20.0 μL; The detection wavelength is λ = 260 nm.

13. The method for extracting five medicinal ingredients from Pueraria root based on a deep eutectic solvent according to claim 11, characterized in that: The HPLC analysis conditions also include gradient elution: 0min: 85%A, 15%B; 16min:70%A,30%B; 30min:100%B,0%A; 31min:85%B,15%A; 40min:85%A,15%B。

Images