Method for extracting chemical components in vietnamese sophora root and method for measuring content of chemical components in vietnamese sophora root

Abstract

The invention belongs to the technical field of natural pharmaceutical chemistry, and particularly relates to a method for extracting chemical components in vietnamese sophora roots and a method for measuring the content of the chemical components in the vietnamese sophora roots. The preparation method comprises the following steps: mixing subprostrate sophora powder and a deep-eutectic solvent, soaking, and performing ultrasonic extraction to obtain a subprostrate sophora powder extracting solution; the radix sophorae tonkinensis powder extracting solution contains at least one of oxymatrine, oxysophocarpine, maackiain and matrine; the eutectic solvent comprises a hydrogen bond donor and a hydrogen bond acceptor; the hydrogen bond donor is ethylene glycol; the hydrogen bond receptor is betaine. The extraction efficiency of the four target compounds in the vietnamese sophora root can be remarkably improved by adopting the deep-eutectic solvent assisted by ultrasonic extraction.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural medicine chemistry, and particularly relates to a method for extracting chemical components from Sophora subprostrata and a method for determining the content of chemical components in Sophora subprostrata. Background Art

[0002] Natural products are an important source for the discovery of innovative drugs. Among the newly marketed drugs, more than one-third are directly or indirectly derived from natural products; and among synthetic drugs, a considerable part also draws on the skeletal structure or pharmacophore of natural products. This shows that the new drug R & D strategy based on natural products still plays a dominant role in modern new drug R & D. However, how to efficiently extract the active ingredients from traditional Chinese medicines has always been the focus of research. Common extraction solvents for traditional Chinese medicines have disadvantages such as high cost, high pollution, high toxicity, flammability, and low extraction rate.

[0003] The traditional Chinese medicine Sophora subprostrata is the dried root and rhizome of Sophora tonkinensis, a plant of the genus Sophora in the family Leguminosae. Sophora subprostrata is also known as Guangdougen. It is cold in nature, bitter in taste, and toxic. It belongs to the lung and stomach meridians and has the effects of clearing heat and detoxifying, relieving sore throat and swelling. It is mainly used to treat symptoms such as fire toxin accumulation, tonsillitis, sore throat, gingival swelling and pain, and oral ulcers. The main chemical components in Sophora subprostrata include flavonoids and alkaloids, such as genistein, sophorone, sophocarpine, matrine, sophocarpine, oxysophocarpine, sophoramine, and sophoranol. However, there is currently no effective method for extracting the chemical components from Sophora subprostrata. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a method for extracting chemical components from Sophora subprostrata and a method for determining the content of chemical components in Sophora subprostrata, and this method has a high extraction rate for the target compounds in Sophora subprostrata.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a method for extracting chemical components from Sophora subprostrata, comprising the following steps:

[0007] Mix Sophora subprostrata powder with a deep eutectic solvent, soak it, and then perform ultrasonic extraction to obtain an extract of Sophora subprostrata powder; at least one of oxymatrine, oxysophocarpine, sophocarpine, and matrine is contained in the extract of Sophora subprostrata powder;

[0008] The deep eutectic solvent includes a hydrogen bond donor and a hydrogen bond acceptor; the hydrogen bond donor is ethylene glycol; the hydrogen bond acceptor is betaine.

[0009] Preferably, the particle size of the Sophora subprostrata powder is 30 - 50 mesh.

[0010] Preferably, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:1 to 5.

[0011] Preferably, the ratio of the dry weight of the sophora flavescens powder to the volume of the deep eutectic solvent is 1 g:(10 - 30) mL.

[0012] Preferably, the time for ultrasonic extraction is 10 - 90 min, the temperature is 20 - 60 °C, and the power is 180 - 420 W.

[0013] Preferably, the water content of the deep eutectic solvent is 0 - 40%.

[0014] The present invention also provides a method for determining the content of chemical components in sophora flavescens, comprising the following steps:

[0015] The extraction solution of the sophora flavescens powder obtained by the method according to the above technical solution is subjected to solid-liquid separation to obtain a sample to be tested;

[0016] The sample to be tested is subjected to high performance liquid chromatography detection, and according to the obtained detection result and the predetermined standard curve, the content of the target compound in the sample to be tested is obtained; according to the mass of the sophora flavescens powder used to prepare the sample to be tested, the content of the target compound in sophora flavescens is calculated;

[0017] The standard curve is a linear curve between the concentration of each target compound and the corresponding liquid chromatography peak area;

[0018] The mobile phase used for the high performance liquid chromatography detection includes mobile phase A and mobile phase B; the flow rate of the mobile phase is 0.8 - 1.0 mL / min; mobile phase A is methanol; mobile phase B is water and diethylamine; the volume concentration of diethylamine in mobile phase B is 0.010 - 0.020%;

[0019] The gradient elution program used for the high performance liquid chromatography detection is as follows: within 0 - 25 min, the volume fraction of mobile phase A increases from 10% to 30%, and the volume fraction of mobile phase B decreases from 90% to 70%; within 25 - 58 min, the volume fraction of mobile phase A increases from 30% to 100%, and the volume fraction of mobile phase B decreases from 70% to 0%;

[0020] The target compounds are oxymatrine, oxysophocarpine, matrine and sophocarpine.

[0021] Preferably, the detector used for the high performance liquid chromatography detection is a diode array detector; the detection wavelength for the high performance liquid chromatography detection is 215 nm.

[0022] Preferably, the chromatographic column used for the high performance liquid chromatography detection is a SHIMSEN Ankylo C18 chromatographic column or waters C18 column; the column temperature for high performance liquid chromatography detection is 30 ± 5 °C, and the injection volume is 10 μL.

[0023] Preferably, after the solid-liquid separation, the following steps are further included: diluting and microfiltrating the liquid obtained from the solid-liquid separation in sequence; the dilution multiple is 3 - 5 times; the pore size of the filter membrane used for microfiltration is 0.2 - 0.25 μm.

[0024] The present invention provides a method for extracting chemical components from Sophora subprostrata, which includes the following steps: mixing Sophora subprostrata powder with a deep eutectic solvent, soaking and then performing ultrasonic extraction to obtain an extraction solution of Sophora subprostrata powder; the extraction solution of Sophora subprostrata powder contains at least one of oxymatrine, oxysophocarpine, maackiain, and matrine; the deep eutectic solvent includes a hydrogen bond donor and a hydrogen bond acceptor; the hydrogen bond donor is ethylene glycol; the hydrogen bond acceptor is betaine. Compared with traditional organic solvents, the deep eutectic solvent (a combination of betaine and ethylene glycol) adopted in the present invention has good polarity, can dissolve both polar and non-polar components simultaneously, can form strong hydrogen bond interactions with target compounds, is beneficial to the extraction of target compounds, and the cavitation effect induced by ultrasonic waves in ultrasonic extraction can enhance the mass transfer between the deep eutectic solvent and Sophora subprostrata, thereby enhancing the extraction efficiency. The method of using a deep eutectic solvent supplemented with ultrasonic extraction in the present invention can significantly improve the extraction efficiency of 4 target compounds in Sophora subprostrata, and the used solvent is safe and the operation is simple.

[0025] The present invention also provides a method for determining the content of chemical components in Sophora subprostrata. The present invention significantly improves the separation of 4 target compounds by controlling the components of the mobile phase, adjusts the elution time, order, and resolution of the target compounds; by controlling the flow rate, it avoids the influence on the elution time and separation effect caused by too fast or too slow flow rate; by adjusting a suitable elution program, it better separates the target compounds from other substances, thereby improving the accuracy of quantification. Description of the Drawings

[0026] Figure 1 It is the standard curve atlas of oxymatrine standard product;

[0027] Figure 2 It is the standard curve atlas of oxysophocarpine standard product;

[0028] Figure 3 It is the standard curve atlas of maackiain standard product;

[0029] Figure 4 It is the standard curve atlas of matrine standard product;

[0030] Figure 5 It is the extraction rate characterization diagram of 4 target compounds of deep eutectic solvents synthesized from different ratios of hydrogen bond acceptors and hydrogen bond donors;

[0031] Figure 6 Characterization diagrams of the extraction rates of 4 target compounds at different ultrasonic powers;

[0032] Figure 7 Characterization diagrams of the extraction rates of 4 target compounds at different ultrasonic temperatures;

[0033] Figure 8 Characterization diagrams of the extraction rates of 4 target compounds at different ultrasonic times;

[0034] Figure 9 Characterization diagrams of the extraction rates of 4 target compounds with eutectic solvents of different water contents;

[0035] Figure 10 Characterization diagrams of the extraction rates of 4 target compounds at different liquid-solid ratios. Detailed implementation manners

[0036] The present invention provides a method for extracting chemical components from Sophora tonkinensis, comprising the following steps:

[0037] Mix Sophora tonkinensis powder and a eutectic solvent, soak and then perform ultrasonic extraction to obtain an extraction solution of Sophora tonkinensis powder; at least one of oxymatrine, oxysophocarpine, maackiain and matrine is contained in the extraction solution of Sophora tonkinensis powder;

[0038] The eutectic solvent includes a hydrogen bond donor and a hydrogen bond acceptor; the hydrogen bond donor is ethylene glycol; the hydrogen bond acceptor is betaine.

[0039] Unless otherwise specified, the present invention has no special requirements for the sources of the raw materials used, and commercially available products well-known to those skilled in the art can be used.

[0040] As an implementation manner, the Sophora tonkinensis powder is a powder dried to a constant weight; the particle size of the Sophora tonkinensis powder is 30-50 mesh, and specifically 40 mesh in specific examples.

[0041] As an implementation manner, the water content of the eutectic solvent is 0-40%, specifically 0%, 10%, 20%, 30% or 40% in specific examples.

[0042] As an implementation manner, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:1-5, specifically 1:1, 1:2, 1:3, 1:4 or 1:5 in specific examples.

[0043] As an implementation manner, the preparation method of the deep eutectic solvent is as follows: mix a hydrogen bond acceptor and a hydrogen bond donor and carry out oil bath heating; the temperature of the oil bath heating is 80-90 °C, specifically 85 °C in specific embodiments; the time of the oil bath heating is 1.5-2.0 h, specifically 2 h in specific embodiments; the oil bath heating is carried out under stirring conditions; the present invention has no special limitation on the stirring rate, and a clear liquid can be obtained.

[0044] As an implementation manner, the ratio of the dry weight of the Sophora subprostrata powder to the volume of the deep eutectic solvent is 1 g:(10-30) mL, specifically 1 g:10 mL, 1 g:15 mL, 1 g:20 mL, 1 g:25 mL or 1 g:30 mL in specific embodiments.

[0045] As an implementation manner, the soaking time is 12-13 h, specifically 12 h in specific embodiments.

[0046] As an implementation manner, the time of ultrasonic extraction is 10-90 min, specifically 10 min, 30 min, 50 min, 70 min or 90 min in specific embodiments, the temperature is 20-60 °C, specifically 20 °C, 30 °C, 40 °C, 50 °C or 60 °C in specific embodiments, and the power is 180-420 W, specifically 180 W, 240 W, 300 W, 360 W or 420 W in specific embodiments.

[0047] In the present invention, oxymatrine, sophocarpine, and matrine are quinolizidine alkaloids, and maackiain is a flavonoid compound.

[0048] In the present invention, the hydrogen bond acceptor is a naturally occurring zwitterionic compound with good solubility and biocompatibility, and the hydrogen bond donor is a polyol with a low melting point and high solubility. The extraction method provided by the present invention has a high extraction rate for 4 compounds in Sophora subprostrata, and the used solvent is safe and the operation is simple.

[0049] The present invention also provides a method for determining the content of chemical components in Sophora subprostrata, comprising the following steps:

[0050] The extraction solution of Sophora subprostrata powder obtained by the method according to the above technical solution is subjected to solid-liquid separation to obtain a sample to be measured;

[0051] The sample to be measured is subjected to high performance liquid chromatography detection, and according to the obtained detection result and a predetermined standard curve, the content of the target compound in the sample to be measured is obtained; according to the mass of the Sophora subprostrata powder used for preparing the sample to be measured, the content of the target compound in Sophora subprostrata is calculated;

[0052] The standard curve is a linear curve between the concentration of each target compound and the corresponding liquid chromatography peak area;

[0053] The mobile phase used in the high performance liquid chromatography (HPLC) detection includes mobile phase A and mobile phase B; the flow rate of the mobile phase is 0.8 - 1.0 mL / min; mobile phase A is methanol; mobile phase B is water and diethylamine; the volume concentration of diethylamine in mobile phase B is 0.010 ～ - 0.020%;

[0054] The gradient elution program used in the HPLC detection is as follows: within 0 - 25 min, the volume fraction of mobile phase A increases from 10% to 30%, and the volume fraction of mobile phase B decreases from 90% to 70%; within 25 - 58 min, the volume fraction of mobile phase A increases from 30% to 100%, and the volume fraction of mobile phase B decreases from 70% to 0%;

[0055] The 4 target compounds are oxymatrine, sophocarpine N-oxide, matrine, and sophocarpetin.

[0056] As an implementation method, the solid-liquid separation is centrifugation; the rotation speed of the centrifugation is 10000 - 12000 rpm, specifically 11000 rpm in the specific example; the time of the centrifugation is 9 - 11 min, specifically 10 min in the specific example.

[0057] As an implementation method, after the solid-liquid separation, it further includes: sequentially diluting and microfiltering the liquid obtained from the solid-liquid separation; the diluent used for dilution is methanol or acetonitrile, specifically methanol in the specific example; the dilution multiple is 3 - 5 times, specifically 4 times in the specific example; the pore size of the filter membrane used for microfiltration is 0.2 - 0.25 μm, specifically 0.22 μm in the specific example; the filter membrane is an organic microporous filter membrane; the material of the organic microporous filter membrane is nylon 6, and the manufacturer is Tianjin Linghang Experimental Equipment Co., Ltd.

[0058] In reverse-phase liquid chromatography, generally chromatographic grade methanol, acetonitrile, and water are used as the mobile phase. In this invention, the mobile phase used is methanol-water. Using pure methanol as the diluent can reduce the solvent effect, and the toxicity of methanol is lower than that of acetonitrile.

[0059] As an implementation method, the HPLC is a reverse-phase high performance liquid chromatography; the detector used in the HPLC detection is a diode array detector (PDA); the detection wavelength for the HPLC detection is 215 nm.

[0060] In this invention, the detection wavelength of the 4 target compounds is all 215 nm because these 4 compounds all have an absorption at 210 nm, but the baseline at 210 nm in the PDA detector is unstable and not easy to quantify.

[0061] As an implementation manner, the chromatographic column used in the high performance liquid chromatography detection is a SHIMSEN Ankylo C18 chromatographic column or a waters C18 column, specifically a SHIMSEN Ankylo C18 chromatographic column in the specific embodiment; the length of the chromatographic column is 150 mm, the inner diameter is 4.6 mm, and the filler particle size is 5 μm; the column temperature for the high performance liquid chromatography detection is 30 ± 5 °C, specifically 30 °C in the specific embodiment, and the injection volume is 10 μL.

[0062] The length and column efficiency of the chromatographic column will affect the peak emergence time and resolution of the target compound. The chromatographic column set in the present invention can effectively separate four compounds and improve the accuracy of quantification. Since the deep eutectic solvent (DES) has a relatively high viscosity, the column temperature set in the present invention can reduce the viscosity, lower the column pressure, and enable better separation of the target compound; the injection volume set in the present invention can avoid the problem that the sample exceeds the detection line due to too large an injection volume, and can also avoid the problem of being affected by baseline fluctuations due to too small an injection volume.

[0063] As an implementation manner, the mobile phase used in the high performance liquid chromatography detection includes mobile phase A and mobile phase B; the flow rate of the mobile phase is 0.8 - 1.0 mL / min, specifically 1.0 mL / min in the specific embodiment; mobile phase A is methanol; mobile phase B is water and diethylamine; the volume concentration of diethylamine in mobile phase B is 0.010 ～ 0.020%, specifically 0.015% in the specific embodiment.

[0064] As an implementation manner, the gradient elution program used in the high performance liquid chromatography detection is as follows: within 0 - 25 min, the volume fraction of mobile phase A increases from 10% to 30%, and the volume fraction of mobile phase B decreases from 90% to 70%; within 25 - 58 min, the volume fraction of mobile phase A increases from 30% to 100%, and the volume fraction of mobile phase B decreases from 70% to 0%.

[0065] The mobile phase can significantly change the separation situation of the target compound. When running the same sample with methanol - water and acetonitrile - water, the peak emergence time, order, resolution, etc. of the target compound will be inconsistent; the above - set flow rate range in the present invention avoids the influence on the peak emergence time and separation effect due to too fast or too slow a flow rate; the elution program set in the present invention can better separate the target compound from other substances, so as to better quantify.

[0066] As an implementation method, the method for drawing the standard curve is as follows: accurately weigh 4 target compounds and add them to chromatographic methanol to prepare a standard mixture solution; gradually dilute the standard mixture solution by 6 concentrations to obtain a standard mixture solution with gradient concentrations; perform high-performance liquid chromatography detection on the standard mixture solution with gradient concentrations to obtain a liquid chromatography, and draw a standard curve according to the linear relationship between the concentration of each target compound and the peak area of the corresponding liquid chromatography.

[0067] In a specific embodiment, the method for drawing the standard curve is as follows: accurately weigh 3 mg of oxymatrine, 0.75 mg of sophocarpine oxide, 0.5 mg of maackiain, and 0.5 mg of matrine, add methanol to prepare a standard mixture solution with oxymatrine at 0.6 mg / mL, sophocarpine oxide at 0.15 mg / mL, maackiain at 0.1 mg / mL, and matrine at 0.1 mg / mL; serially dilute the standard mixture solution to obtain a standard mixture solution with gradient concentrations. Among them, the gradient concentrations of oxymatrine are: 9.375, 18.75, 37.5, 75, 150, 300, 600 μg / mL, the gradient concentrations of sophocarpine oxide are: 2.343, 4.688, 9.375, 18.75, 37.5, 75, 150 μg / mL, and the gradient concentrations of maackiain and matrine are both: 1.563, 3.125, 6.25, 12.5, 25, 50, 100 μg / mL; perform high-performance liquid chromatography detection on the standard mixture solution with gradient concentrations to obtain a liquid chromatography; perform linear regression with the peak area of the liquid chromatography as the ordinate and the concentration of each target compound in the standard mixture solution as the abscissa to obtain a regression equation, and draw a standard curve.

[0068] As an implementation method, the regression equation of oxymatrine is Y = 10000000X + 57587, R is 0.9998; the regression equation of sophocarpine oxide is Y = 20000000X - 25573, R is 0.9997; the regression equation of maackiain is Y = 50000000X + 52792, R is 0.9998; the regression equation of matrine is Y = 20000000X + 24703, R is 0.9997.

[0069] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention, but they cannot be understood as limiting the protection scope of the present invention.

[0070] Example 1

[0071] Mix betaine and ethylene glycol in a molar ratio of 1:2, heat in an oil bath at 85 °C for 2 h until a clear solution is obtained, and continuously stir during heating to ensure uniform mixing of the solution to obtain a deep eutectic solvent DES-10 with a water content of 20%;

[0072] Weigh 0.1 g of Sophora flavescens powder (40 mesh), place it in a 7 mL centrifuge tube, transfer the above-mentioned deep eutectic solvent DES-10, add it according to the liquid-solid ratio of 20 mL / g, mix evenly, soak for 12 h, ultrasonically extract at 30 °C for 30 min at 300 W. Centrifuge the obtained Sophora flavescens powder extract at 11000 rpm for 10 min, take the supernatant, dilute it 4 times with methanol, filter it through a 0.22 μm microporous filter membrane (nylon 6, Tianjin Linghang Experimental Equipment Co., Ltd.), and finally quantitatively analyze 4 target compounds by high performance liquid chromatography (HPLC). The specific quantitative method is shown in the performance test section.

[0073] Comparative Examples 1-20

[0074] The difference from Example 1 is that the deep eutectic solvent DES-10 in Example 1 is respectively replaced by deep eutectic solvents DES-1-DES-9, DES-11-DES-18, methanol, 95 wt% ethanol and water, and the rest is the same as Example 1.

[0075] The component ratios and water contents of the deep eutectic solvents DES-1-9, DES-11-18 are shown in Table 2.

[0076] Comparative Example 21

[0077] The difference from Example 1 is that ultrasonic extraction is not carried out, and directly extract with the deep eutectic solvent DES-10, and the rest is the same as Example 1.

[0078] Performance Test

[0079] (1) Preparation of Standard Mixture Solution

[0080] Accurately weigh 3 mg of oxymatrine, 0.75 mg of sophocarpine, 0.5 mg of maackiain and 0.5 mg of matrine, and prepare a standard mixture solution of oxymatrine 0.6 mg / mL, sophocarpine 0.15 mg / mL, maackiain 0.1 mg / mL and matrine 0.1 mg / mL with methanol.

[0081] (2) HPLC-PDA Content Determination Chromatographic Conditions

[0082] Chromatographic column: SHIMSEN Ankylo C18 chromatographic column (4.6×150 mm, 5 μm); flow rate was 1.0 mL / min; mobile phase A was methanol; mobile phase B was water and diethylamine, and the volume concentration of diethylamine in mobile phase B was 0.015%; gradient elution program was as follows: within 0 - 25 min, the volume fraction of mobile phase A increased from 10% to 30%, and the volume fraction of mobile phase B decreased from 90% to 70%; within 25 - 58 min, the volume fraction of mobile phase A increased from 30% to 100%, and the volume fraction of mobile phase B decreased from 70% to 0%; detection wavelength was 215 nm; column temperature was 30 °C, and injection volume was 10 μL.

[0083] (3) Investigation on the linear range of HPLC - PDA content determination

[0084] Dilute the standard product mixed solution in (2) by half - fold to obtain standard product mixed solutions with gradient concentrations. The gradient concentrations of oxymatrine were: 9.375, 18.75, 37.5, 75, 150, 300, 600 μg / mL; the gradient concentrations of sophocarpine were: 2.343, 4.688, 9.375, 18.75, 37.5, 75, 150 μg / mL; the gradient concentrations of maackiain and matrine were both: 1.563, 3.125, 6.25, 12.5, 25, 50, 100 μg / mL; Determine according to the chromatographic conditions in (3). With the peak area of the chromatographic peak as the ordinate (Y) and the concentration of each component in the mixed standard product as the abscissa (X), perform linear regression to obtain the regression equation. The results are shown in Table 1, and the standard curves of the 4 compounds are shown in Figures 1 to 4 .

[0085] Table 1 Results of the investigation on the linear relationship of 4 target compounds

[0086]

[0087] (4) Preparation and screening of deep eutectic solvents

[0088] 1) Preparation of deep eutectic solvents

[0089] Select 18 kinds of deep eutectic solvents, namely choline chloride - ethylene glycol, choline chloride - 1,2 - propanediol, choline chloride - glycerol, choline chloride - xylitol, choline chloride - p - toluenesulfonic acid, choline chloride - 1,3 - benzenediol, choline chloride - urea, choline chloride - fructose, choline chloride - glucose, betaine - ethylene glycol, betaine - xylitol, betaine - glycerol, menthol - formic acid, menthol - 1,3 - butanediol, betaine - glycerol - glucose, menthol - levulinic acid - 1,3 - butanediol, menthol - levulinic acid - tert - butanol, and menthol - 1,3 - butanediol - tert - butanol for preparation. The hydrogen bond acceptor (HBA), hydrogen bond donor (HBD), and water are mixed according to the ratios in Table 2 respectively. Heat in an oil bath at 85 °C for 2 h until a clear solution is obtained. Stir continuously during heating to ensure uniform mixing of the solution.

[0090] Table 2 Different types of deep eutectic solvents

[0091]

[0092]

[0093] 2) Screening of deep eutectic solvents

[0094] To investigate the extraction rates of 4 target compounds in Sophora tonkinensis by the above different types of deep eutectic solvents and select the best deep eutectic solvent, weigh 0.1 g of Sophora tonkinensis powder and place it in a 7 - mL centrifuge tube. Pipette the above 18 kinds of deep eutectic solvents and traditional solvents (water, 95 wt% ethanol, methanol) respectively, add them according to the liquid - solid ratio of 20 mL / g, mix evenly, soak for 12 h, ultrasonically extract at 30 °C with 300 W for 30 min. Centrifuge the obtained extraction solution of Sophora tonkinensis powder at 11000 rpm for 10 min, take the supernatant, dilute it 4 times with methanol, filter through a 0.22 - μm microporous membrane, and finally quantitatively analyze the 4 target compounds by high - performance liquid chromatography (HPLC), calculate the extraction rate, and screen out the best solvent. The results are shown in Table 3.

[0095] Table 3 Extraction rates of 4 target compounds in Sophora tonkinensis powder by different solvents

[0096]

[0097]

[0098] As can be seen from Table 3, the extraction efficiency of DES-10 is higher than that of other solvents. Perhaps the DES composed of betaine and ethylene glycol exhibits moderate polarity, enabling it to dissolve both polar and non-polar components simultaneously. When this DES system is used to extract the target compounds from Sophora subprostrata, it can adapt to target compounds with different polarities, thereby improving the extraction efficiency. Compared with traditional solvents, perhaps the ability of DES-10 to form hydrogen bonds with target compounds is stronger than that of traditional solvents. Moreover, ultrasonic extraction can help improve the extraction efficiency of DES-10 for target compounds.

[0099] 3) Single-factor experiments

[0100] Using the deep eutectic solvent (DES-10) screened in the above-mentioned "2) Screening of deep eutectic solvents" as the extraction solvent, single-factor experiments were carried out based on its specific steps as the basic experimental steps.

[0101] Figure 5 It is a characterization diagram of the extraction rates of 4 target compounds by deep eutectic solvents (DES-10) synthesized from hydrogen bond acceptors and hydrogen bond donors in different ratios (molar ratio: 1:1, 1:2, 1:3, 1:4, 1:5). Adjusting the molar ratio will affect the physical and chemical properties of DES. As Figure 5 can be seen, under the conditions of the molar ratio of hydrogen bond acceptor (betaine) and hydrogen bond donor (ethylene glycol) defined in the present invention, all 4 target compounds show good extraction effects. Among them, as the proportion of ethylene glycol increases, the extraction efficiency gradually increases. Perhaps it is because the viscosity of DES decreases, which is beneficial to the release of target compounds into DES. When the molar ratio of hydrogen bond acceptor (betaine) and hydrogen bond donor (ethylene glycol) reaches 1:4, the extraction efficiency is the highest. However, as the molar ratio further increases, the extraction efficiency decreases slightly. Perhaps it is because the decrease in the proportion of betaine limits the hydrogen bond interaction between the target compounds and DES.

[0102] Figure 6 It is a characterization diagram of the extraction rates of 4 target compounds at different ultrasonic powers (180, 240, 300, 360, 420 W). As Figure 6 can be seen, under the extraction power conditions defined in the present invention, all 4 target compounds show good extraction effects. As the ultrasonic power increases, the cavitation effect induced by ultrasonic waves enhances, the mass transfer between the solvent and the plant matrix enhances, and the extraction efficiency increases. When the ultrasonic power is 360 W, the extraction efficiency is the highest. But as the power continues to increase, the extraction efficiency decreases slightly. Perhaps too high power leads to excessive damage to the cell structure, which is instead not conducive to the release and dissolution of active ingredients.

[0103] Figure 7 It is a characterization diagram of the extraction rates of 4 target compounds at different ultrasonic temperatures (20, 30, 40, 50, 60 °C). As Figure 7It can be seen that under the extraction temperature conditions defined in the present invention, all 4 target compounds exhibit good extraction effects. As the temperature increases, the extraction efficiency slightly increases. When the extraction temperature is 30 °C, the extraction efficiency is the highest. As the temperature continues to rise, the extraction efficiency basically remains unchanged. This may be because the 4 selected target compounds are relatively stable at 20-60 °C and are not easily decomposed by temperature.

[0104] Figure 8 It is a characterization diagram of the extraction rates of 4 target compounds under different ultrasonic times (10, 30, 50, 70, 90 min). From Figure 8 It can be seen that under the ultrasonic time conditions defined in the present invention, all 4 target compounds exhibit good extraction effects. As the ultrasonic time increases, the extraction efficiency increases. Among them, when the extraction time is 30 min, the extraction efficiency is the highest. Prolonging the ultrasonic time can optimize the polarity and fluidity of the solvent, thereby improving the extraction efficiency. However, an overly long ultrasonic time may cause the solvent temperature to rise, affecting the polarity matching of the solvent, and thus reducing the extraction efficiency.

[0105] Figure 9 It is a characterization diagram of the extraction rates of 4 target compounds by deep eutectic solvents with different water contents (0, 10, 20, 30, 40%). From Figure 9 It can be seen that under the conditions of the water content of the extraction solvent (DES-10) defined in the present invention, all 4 target compounds exhibit good extraction effects. As the water content increases, the viscosity of DES decreases, enhancing its fluidity, thereby improving the extraction efficiency; while excessive water will disrupt the hydrogen bond network of DES, reducing its interaction with the target components, resulting in a decrease in the extraction efficiency. Among them, when the water content is 30%, the extraction efficiency is the highest.

[0106] Figure 10 It is a characterization diagram of the extraction rates of 4 target compounds under different liquid-solid ratios (10, 15, 20, 25, 30 mL / g). From Figure 10 It can be seen that under the conditions of the dosages of Sophora subprostrata powder and deep eutectic solvent defined in the present invention, all 4 target compounds exhibit good extraction effects. An appropriate liquid-solid ratio can optimize the physical and chemical properties of the solvent, improve the mass transfer efficiency, and thus improve the extraction efficiency. However, an excessively high liquid-solid ratio will dilute the solvent, reducing its interaction with the target compounds, resulting in a decrease in the extraction efficiency. When the liquid-solid ratio is 25 mL / g, the extraction efficiency is the highest.

[0107] As can be seen from the above embodiments, compared with traditional organic solvents, the deep eutectic solvent (DES-10) provided by the present invention can significantly improve the extraction efficiency of four target compounds (oxymatrine, oxysophocarpine, maackiain, and matrine) from Sophora subprostrata. The present invention uses a deep eutectic solvent supplemented with ultrasonic extraction to extract Sophora subprostrata powder. Since the deep eutectic solvent forms a strong hydrogen bond with the target compounds, it is beneficial to the extraction of the target compounds. At the same time, the ultrasonic-assisted extraction conditions are optimized, thereby improving the extraction rate of the four target compounds in Sophora subprostrata powder.

[0108] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for extracting chemical components from Sophora flavescens, characterized in that: The following steps are involved: The sophora flavescens powder and the low eutectic solvent are mixed, and ultrasonic extraction is performed after soaking to obtain a sophora flavescens powder extract; the sophora flavescens powder extract contains at least one of oxymatrine, oxysophocarpine, sophora flavescens and matrine; The low eutectic solvent comprises a hydrogen bond donor and a hydrogen bond acceptor; the hydrogen bond donor is ethylene glycol; and the hydrogen bond acceptor is betaine.

2. The method according to claim 1, characterized in that The particle size of the Sophora flavescens powder is 30 to 50 meshes.

3. The method according to claim 1, characterized in that The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is 1:1-5.

4. The method according to claim 1 or 3, characterized in that: The ratio of the dry weight of the Sophora flavescens powder to the volume of the low eutectic solvent is 1 g: (10-30) mL.

5. The method according to claim 1, characterized in that The ultrasonic extraction time is 10 to 90 minutes, the temperature is 20 to 60° C., and the power is 180 to 420W.

6. The method according to claim 1 or 3, characterized in that: The water content of the deep eutectic solvent is 0-40%.

7. A method for determining the content of chemical components in Radix Sophorae Tonkinensis, characterized in that: The following steps are involved: The extract of Sophora flavescens powder obtained by the method according to any one of claims 1 to 6 is subjected to solid-liquid separation to obtain a sample to be tested; The sample to be tested is subjected to high performance liquid chromatography detection, and the content of the target compound in the sample to be tested is obtained according to the obtained detection result and a predetermined standard curve; the content of the target compound in the Radix Sophorae Tonkinensis is calculated according to the mass of the Radix Sophorae Tonkinensis powder used to prepare the sample to be tested; The standard curve is a linear curve between the concentration of each target compound and the corresponding liquid chromatography peak area; The mobile phase used for the high performance liquid chromatography detection includes mobile phase A and mobile phase B; the flow rate of the mobile phase is 0.8-1.0 mL / min; the mobile phase A is methanol; the mobile phase B is water and diethylamine; the volume concentration of diethylamine in the mobile phase B is 0.010 ～ 0.020%; The gradient elution program used in the HPLC detection is: within 0 to 25 minutes, the volume fraction of mobile phase A increases from 10% to 30%, and the volume fraction of mobile phase B decreases from 90% to 70%; within 25 to 58 minutes, the volume fraction of mobile phase A increases from 30% to 100%, and the volume fraction of mobile phase B decreases from 70% to 0%; The target compounds are oxymatrine, oxysophocarpine, matrine and sophora flavescens.

8. The method according to claim 7, characterized in that The detector used for the high performance liquid chromatography detection is a diode array detector; the detection wavelength of the high performance liquid chromatography detection is 215nm.

9. The method according to claim 7, characterized in that: The chromatographic column used for the high performance liquid chromatography detection is a SHIMSEN Ankylo C18 chromatographic column or a waters C18 column; the column temperature of the HPLC detection is 30±5°C, and the injection volume is 10 μL.

10. The method according to claim 7, characterized in that After the solid-liquid separation, the method further includes: diluting and microfiltering the liquid obtained by the solid-liquid separation in sequence; the dilution multiple is 3 to 5 times; and the pore size of the filter membrane used for the microfiltration is 0.2 to 0.25 μm.

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