Radix paeoniae alba extract as well as extraction method and application thereof

Abstract

The invention discloses a radix paeoniae alba extract as well as an extraction method and application thereof, and belongs to the technical field of active substance extraction. The method comprises the following steps: synthesizing a hydrophilic eutectic solvent from L-proline and glycerol; synthesizing a lipophilic eutectic solvent from betaine and benzyl alcohol; the preparation method comprises the following steps: synthesizing a hydrophilic eutectic solvent, a lipophilic eutectic solvent and deionized water into a double-phase eutectic solvent system; and adding the white peony root powder into a double-phase eutectic solvent system, carrying out ultrasonic-assisted extraction, and purifying with an AB-8 macroporous adsorption resin chromatographic column to obtain the white peony root extract. According to the radix paeoniae alba extract and the extraction method and application thereof, the extraction efficiency and purity of the radix paeoniae alba extract are improved, and the radix paeoniae alba extract has a remarkable neuron protection effect and can effectively relieve nerve injury and protect nerve cells.

Description

Technical Field

[0001] The invention belongs to the technical field of active substance extraction, and specifically relates to a white peony root extract and an extraction method and application thereof. Background Art

[0002] Ischemic stroke is caused by thrombosis in cerebral blood vessels, which interrupts blood flow to the brain, causing neurons to become functionally impaired due to lack of energy and oxygen. During the process of blood vessel recanalization and blood flow restoration, part of the ischemic brain tissue may suffer further damage, exacerbating neurological dysfunction.

[0003] At present, the main clinical treatment is thrombolytic therapy, including the use of tissue plasminogen activator thrombolysis and mechanical thrombectomy. However, there is still a lack of effective treatment for damage caused by vascular recanalization and blood flow restoration. In this context, natural active substances have shown great potential in protecting neurons in ischemic stroke. Studies have shown that natural active substances extracted from traditional Chinese medicinal materials can protect neurons by reducing oxidative stress, reducing inflammatory responses, reducing neuronal apoptosis and autophagy, and enhancing the repair ability of neurons.

[0004] White peony root, its active ingredients such as total glycosides of paeoniflorin have been confirmed by modern research to have the effect of protecting neurons. The pharmacological effects of white peony root include anti-inflammatory, analgesic, and liver protection, and have significant effects on the nervous system, skeletal muscle, etc. White peony root total glycosides capsule has an antioxidant effect, can scavenge free radicals, reduce oxidative stress, protect neurons from damage, and has application prospects in neural repair. In addition, white peony root total glycosides capsule can also reduce brain edema, improve brain tissue microenvironment, and promote brain tissue repair. However, traditional extraction methods have problems such as low extraction efficiency, strong toxicity, and difficulty in biodegradation, which have a negative impact on the environment and human health, limiting its application. Therefore, developing new extraction technologies and methods to improve the extraction efficiency and bioavailability of active ingredients in white peony root and reduce the impact on the environment and human body is of great significance for the extraction of natural active substances. Summary of the invention

[0005] The present invention aims to provide a white peony extract and an extraction method and application thereof, which improves the extraction efficiency and purity of the white peony extract, and the white peony extract has a significant effect on neuron protection, can effectively reduce nerve damage and protect nerve cells.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for extracting white peony root extract comprises the following steps:

[0008] S1. Evenly mix L-proline and glycerol, heat and stir in an oil bath until the mixture is transparent and uniform, to obtain a hydrophilic deep eutectic solvent;

[0009] S2, mixing betaine and benzyl alcohol uniformly, heating and stirring in an oil bath until the mixture is transparent and uniform, to obtain a lipophilic deep eutectic solvent;

[0010] S3, the hydrophilic deep eutectic solvent obtained in step S1 and the lipophilic deep eutectic solvent obtained in step S2 are uniformly mixed in a certain volume ratio, deionized water is added, and the mixture is heated and stirred in an oil bath to obtain a two-phase deep eutectic solvent system;

[0011] S4, adding the white peony root powder to the biphasic deep eutectic solvent system obtained in step S3, performing ultrasound-assisted extraction, filtering the filtrate, centrifuging, and discarding the precipitate to obtain a white peony root extract;

[0012] S5. Load the white peony root extract obtained in step S4 onto an AB-8 macroporous adsorption resin chromatography column, elute with ethanol, collect the eluate, concentrate, and vacuum dry to obtain the white peony root extract.

[0013] Preferably, in step S1, L-proline and glycerol are uniformly mixed in a molar ratio of 1:3, and heated in an oil bath at 80° C. with stirring until the mixture is transparent and uniform, thereby obtaining a hydrophilic low eutectic solvent.

[0014] Preferably, in step S2, betaine and benzyl alcohol are mixed uniformly in a molar ratio of 1:3, and heated in an oil bath at 80° C. with stirring until the mixture is transparent and uniform, thereby obtaining a lipophilic deep eutectic solvent.

[0015] Preferably, in step S3, the hydrophilic deep eutectic solvent obtained in step S1 and the lipophilic deep eutectic solvent obtained in step S2 are uniformly mixed in a volume ratio of 7:3, 50%-60% volume of deionized water is added, and the mixture is heated and stirred in an oil bath at 80° C. for 60 min to obtain a two-phase deep eutectic solvent system.

[0016] Preferably, in step S4, the white peony root powder is obtained by crushing the white peony root and passing it through a 20-60 mesh sieve.

[0017] Preferably, in step S4, the white peony root powder is added to the two-phase low eutectic solvent system obtained in step S3 at a liquid-to-solid ratio of 45 to 50 mL / g.

[0018] Preferably, in step S4, the ultrasonic power is 200-250 W, the extraction temperature is 50-60° C., the extraction time is 25-30 min, the centrifugal speed is 5000-10000 r / min, and the centrifugal time is 50-60 min.

[0019] Preferably, in step S5, the white peony extract obtained in step S4 is loaded onto an AB-8 macroporous adsorption resin chromatography column and eluted with ethanol, the loading volume is 0.1-0.2BV, the elution flow rate is 1.5BV / h, and each gradient elution is 3-5BV.

[0020] Preferably, in step S5, the vacuum drying temperature is 50-60°C, the drying time is 100-120 min, and the vacuum degree is 60-80 Pa.

[0021] The present invention also provides a white peony root extract obtained by the extraction method.

[0022] The present invention also provides the white peony root extract obtained by the extraction method or the application of the white peony root extract in preparing a neuron protection drug.

[0023] The present invention also provides a pharmaceutical composition comprising one or more white peony root extracts.

[0024] The pharmaceutical composition comprises the white peony root extract as an active ingredient and a pharmaceutically acceptable carrier.

[0025] A pharmaceutical preparation comprises a therapeutically effective amount of any one of the white peony root extracts described above and a pharmaceutically acceptable excipient.

[0026] The pharmaceutical preparations include the following dosage forms: oral preparations (such as tablets, capsules, solutions or suspensions); injectable preparations (such as injectable solutions or suspensions, or injectable dry powders, which can be used immediately by adding injection water before injection) and local preparations (such as ointments or solutions).

[0027] The carrier used in the pharmaceutical composition of the present invention is a common carrier available in the pharmaceutical field, including: binders, lubricants, disintegrants, solubilizers, diluents, stabilizers, suspending agents, pigments, flavoring agents, etc. for oral preparations; preservatives, solubilizers, stabilizers, etc. for injectable preparations; bases, diluents, lubricants, preservatives, etc. for local preparations. The pharmaceutical preparations can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically), and if some drugs are unstable under gastric conditions, they can be formulated into enteric-coated tablets.

[0028] Compared with the prior art, the present invention has the following advantages and technical effects:

[0029] The present invention discloses a white peony root extract and an extraction method and application thereof. The used dual-phase deep eutectic solvent system is low in cost, simple to prepare, non-toxic and harmless, and meets the requirements of environmental protection and economy. The extraction method improves the extraction efficiency of active ingredients in white peony root, effectively enriches the active ingredients in white peony root, and improves the purity and biological activity of the extract. It has been verified by in vitro and in vivo experiments that the obtained white peony root extract shows a significant neuroprotective effect, can effectively reduce nerve damage and protect nerve cells, and has important application potential for the prevention and treatment of neurological diseases.

[0030] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a single factor experimental diagram for the optimization of the extraction process of Examples 1-29 of the present invention, wherein: Figure 1 A in the figure is the viability of PC12 cells in the OGD / R model pretreated with extracts at different extraction times. Figure 1 B in the figure is the viability of PC12 cells in the OGD / R model pretreated with extracts at different liquid-to-solid ratios. Figure 1 C in the figure is the viability of PC12 cells in the OGD / R model pretreated with extracts at different extraction temperatures. Figure 1 D in the figure is the viability of PC12 cells in the OGD / R model pretreated with extracts at different ultrasonic powers;

[0032] Figure 2 It is a response surface contour map of the extraction process optimization of Examples 1-29 of the present invention, wherein, Figure 2 A in the figure is the extraction time X 1 , liquid-to-solid ratio X 2 Effects on the viability of PC12 cells in the OGD / R model, Figure 2 B in the figure is the extraction time X 1 , extraction temperature X 3 Effects on the viability of PC12 cells in the OGD / R model, Figure 2 C is the extraction time X 1 , Ultrasonic power X 4 Effects on the viability of PC12 cells in the OGD / R model, Figure 2 D is the liquid-to-solid ratio X 2 , extraction temperature X 3 Effects on the viability of PC12 cells in the OGD / R model, Figure 2 E is the liquid-to-solid ratio X 2 , Ultrasonic power X 4 Effects on cell viability, Figure 2 F is the extraction temperature X 3 , Ultrasonic power X 4Effects on cell viability;

[0033] Figure 3 It is a three-dimensional response surface diagram of the extraction process optimization of Examples 1-29 of the present invention, wherein: Figure 3 A in the figure is the extraction time X 1 , liquid-to-solid ratio X 2 Effects on the viability of PC12 cells in the OGD / R model, Figure 3 B in the figure is the extraction time X 1 , extraction temperature X 3 Effects on the viability of PC12 cells in the OGD / R model, Figure 3 C is the extraction time X 1 , Ultrasonic power X 4 Effects on the viability of PC12 cells in the OGD / R model, Figure 3 D is the liquid-to-solid ratio X 2 , extraction temperature X 3 Effects on the viability of PC12 cells in the OGD / R model, Figure 3 E is the liquid-to-solid ratio X 2 , Ultrasonic power X 4 Effects on the viability of PC12 cells in the OGD / R model, Figure 3 F is the extraction temperature X 3 , Ultrasonic power X 4 Effects on the viability of PC12 cells in the OGD / R model;

[0034] Figure 4 The protective effect of the white peony root extract provided in Example 30 on PC12 cells in the OGD / R model;

[0035] Figure 5 The neuroprotective effect of the white peony root extract on MCAO mice provided in Example 30; wherein, Figure 5 A in the figure is the design of the mouse MCAO experiment. Figure 5 B is a representative photograph of five consecutive coronal brain sections taken by TTC staining, with the infarct area indicated in white. Figure 5 C in the figure is the quantitative analysis of cerebral infarction volume. Figure 5 D in the figure is the modified mNSS score. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0037] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0038] White peony root is the dried root of Paeonia lactiflora Pall., a plant of the genus Paeonia in the family Paeoniaceae, and was purchased from the Bozhou Chinese herbal medicine wholesale market in Anhui Province.

[0039] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in the art and can be purchased through commercial channels.

[0040] Embodiment 1 This embodiment provides a white peony root extract, and the extraction method comprises the following steps:

[0041] S1. L-proline and glycerol were mixed uniformly in a molar ratio of 1:3, and heated in an oil bath at 80° C. with stirring until the mixture was transparent and uniform, to obtain a hydrophilic deep eutectic solvent;

[0042] S2, mixing betaine and benzyl alcohol in a molar ratio of 1:3, heating in an oil bath at 80° C. with stirring until the mixture is transparent and uniform, to obtain a lipophilic deep eutectic solvent;

[0043] S3, the hydrophilic deep eutectic solvent obtained in step S1 and the lipophilic deep eutectic solvent obtained in step S2 are uniformly mixed in a volume ratio of 7:3, 60% volume of deionized water is added, and the mixture is heated and stirred in an oil bath at 80° C. for 60 min to obtain a two-phase deep eutectic solvent system;

[0044] S4, the white peony powder (white peony powder is crushed and passed through a 60-mesh sieve) is added to the two-phase low eutectic solvent system obtained in step S3 at a liquid-to-solid ratio of 50 mL / g, and ultrasonic-assisted extraction is performed, wherein the ultrasonic power is 250 W, the extraction temperature is 50 ° C, the extraction time is 30 min, the filtrate is filtered, and the filtrate is centrifuged at 5000 r / min for 60 min, and the precipitate is discarded to obtain the white peony extract.

[0045] Example 2-Example 25, the extraction method of white peony root extract is the same as that of Example 1, except that in step S4, the solid-liquid ratio, ultrasonic power, extraction temperature and extraction time, the specific parameters are shown in Table 1.

[0046] Table 1 Extraction process parameters of white peony root extract

[0047]

[0048]

[0049] In Examples 26-29, the reaction parameters were completely consistent with those in Example 25 above, and a center point test was performed.

[0050] The effects of the white peony root extracts provided in Examples 1-29 were verified by the following experiments.

[0051] The experimental scheme is as follows: PC12 cells were cultured and passaged in DMEM medium containing 4.5g / L glucose, 10% fetal bovine serum, and 1% penicillin / streptomycin. The white peony extract was diluted 1600 times with DMEM medium and pretreated with PC12 cells for 2 hours. The original medium was then discarded, and the culture medium was replaced with sugar-free Neurobasal-A medium, and the cells were placed in an oxygen-deficient environment for 4 hours of hypoxic and glucose-deficient treatment. After hypoxic treatment, the cells were placed back under normal conditions for reoxygenation and resugar treatment to simulate the reperfusion process after ischemia, and culture was continued for 24 hours. Finally, 10μL of CCK8 reagent was added to each well and incubated in the incubator for 1-2 hours. The OD value was detected at 450nm on an enzyme reader. The cell viability was calculated using the OD value of the control group as a reference. The results are shown in Tables 2 and Figure 1 .

[0052] Table 2 Cell viability results

[0053]

[0054]

[0055]

[0056] As shown in Table 2, the white peony extract provided in Examples 1-29 has an effect on the viability of PC12 cells in the OGD / R model.

[0057] Depend on Figure 1 It can be seen that the single factor experimental results of the extraction process optimization of Examples 1-29 are that the optimal extraction time is 20 min, the optimal liquid-to-solid ratio is 40 mL / g, the optimal extraction temperature is 50° C., and the optimal ultrasonic power is 250 W.

[0058] According to the results of the single factor experiment, the optimal condition in the single factor experiment was set as the 0 level, and the two conditions before and after the optimal condition were -1 and 1 levels respectively. Using Design-Expert 13.0.5.0 software, the Box-Behnken (BBD) in the response surface was used to extract the time X 1 , liquid-to-solid ratio X 2 , extraction temperature X 3 and ultrasonic power X 4 The above response surface optimization experiments of Examples 1-29 were designed with cell viability as the independent variable and response value Y as the cell viability.

[0059] Y=81.3100+0.2900X 1 +0.3208X 2 +0.2625X 3 -0.3083X 4 +0.4350X 1

[0060] X 2 +0.0925X 1 X 3 -0.2075X 1 X 4 +0.0225X 2 X 3 +0.1800X 2 X 4 -0.2125X 3 X 4 -1.3200X 1 2 -1.0800X 2 2 -1.6900X 3 2 -1.1900X 4 2

[0061] The results are shown in Table 3.

[0062] Table 3 ANOVA results of the response surface quadratic model for the optimization of the extraction process of white peony root extract

[0063]

[0064]

[0065] Note: * indicates P < 0.05, the effect is significant; ** indicates P < 0.01, the effect is very significant; *** indicates P < 0.001, the effect is extremely significant.

[0066] As shown in Table 3, the P value of the lack of fit term>0.05 is not significant, indicating that the model is suitable for the extraction of white peony root, and the correlation coefficient R 2 is 0.9541, Adj R 2 With Pred R 2 The difference was less than 0.2, and the coefficient of variation CV% was 0.4446% <10%, indicating that the regression model was feasible within the range of variables and had high experimental reliability and precision.

[0067] X 1 2 , X 2 2 , X 3 2 and X 4 2 The effect on the extraction of white peony root extract was extremely significant (P<0.001); 2 and X 4The effect on it was very significant (P<0.01). The results showed that the model was extremely significant (P<0.001), and the lack of fitting terms was not significant (P=0.2945>0.05), indicating that the model can better analyze and predict the effects of various extraction conditions on white peony extract.

[0068] Response surface contour plots and response surface 3D plots are shown in Figure 2. Figure 2 and Figure 3 .

[0069] Depend on Figure 2 and Figure 3 It can be seen that when the slope of the response surface 3D graph is larger and the response surface contour graph is closer to an ellipse, it means that the interaction between the two factors has a more obvious effect on the extraction effect of white peony extract. The experimental results show that the interaction between extraction time and liquid-to-solid ratio has a significant effect on the extraction effect of white peony extract.

[0070] The optimal value of each variable was obtained by using Design-Expert Software. The theoretical optimal extraction process was extraction time 29.69min, liquid-to-solid ratio 47.19mL / g, extraction temperature 56.35℃ and ultrasonic power 200.62W, and the predicted cell survival rate was 79.22%. Considering the actual operation, the conditions were modified to extraction time 30min, liquid-to-solid ratio 47mL / g, extraction temperature 56℃ and ultrasonic power 200W, and the actual cell survival rate was measured to be 79.32±0.46% (n=3). The actual value was close to the theoretical predicted value, indicating that the extraction process conditions of white peony extract based on biphasic low eutectic solvent obtained by the design model were stable and reliable.

[0071] Embodiment 30 This embodiment provides a white peony root extract, and the extraction process comprises the following steps:

[0072] S1-S4 are the same as in Example 1.

[0073] S5. Load the white peony root extract obtained in step S4 onto an AB-8 macroporous adsorption resin chromatography column and elute with ethanol, wherein the loading volume is 0.1 BV, the elution flow rate is 1.5 BV / h, and each gradient elution is 5 BV. Collect the eluate, concentrate it, and vacuum dry it at 50° C. for 120 min at a vacuum degree of 80 Pa to obtain the white peony root extract.

[0074] The effect of the white peony root extract provided in Example 30 was verified by the following test.

[0075] 1. Determination of the content of total peony glucosides in white peony extract. The test scheme is as follows: accurately weigh 0.1g white peony extract, add 0.5mL of 5% NaOH solution, hydrolyze in boiling water bath for 2h, then dilute to 5mL volumetric flask with 40% analytical methanol, and filter with 0.45μm filter membrane. Benzoic acid is used as reference. Chromatographic model and conditions: HPLC system uses Dalian Yiteli Supersil ODS2 (5μm, 4.6mm×250mm). The mobile phase is: 0.1% phosphoric acid aqueous solution (phase A), chromatographic pure acetonitrile (phase B), gradient elution, injection volume 10μL, column oven set at 35℃, flow rate set at 1mL / min, detection wavelength 230nm. The determination results show that the content of total peony glucosides in white peony extract is 90.27±2.53mg / g (n=3).

[0076] 2. Evaluation of the neuroprotective effect of white peony root extract in vitro

[0077] The test plan is as follows:

[0078] PC12 cell culture: PC12 cells were cultured and passaged in DMEM medium containing 4.5 g / L glucose, 10% fetal bovine serum, and 1% penicillin / streptomycin.

[0079] Construction of OGD / R model of PC12 cells and experimental grouping: First, oxygen-glucose deprivation treatment was performed, the culture medium was replaced with sugar-free Neurobasal-A medium, and the cells were placed in an oxygen-deficient environment for 4 hours of hypoxia and glucose deprivation. Then, reoxygenation and resugar treatment was performed, and the cells were placed under normal conditions for reoxygenation and resugar treatment to simulate the reperfusion process after ischemia, and culture was continued for 24 hours. The experiment was divided into 4 groups: blank group (Ctrl), model group (OGD / R), low-dose group of white peony extract (10μg / mL), and high-dose group of white peony extract (20μg / mL). White peony extract was dissolved in dimethyl sulfoxide (DMSO) to an initial concentration of 1mg / mL, and then diluted to each final concentration using DMEM medium.

[0080] CCK8 method to detect cell viability: PC12 cells treated with drug for 2 hours were subjected to OGD / R modeling and cultured for 24 hours. Finally, 10 μL of CCK8 reagent was added to each well and incubated in the incubator for 1-2 hours. The OD value was detected at 450nm on an ELISA reader. The OD value of the control group was used as a reference to calculate cell viability. The results are shown in Figure 4 .

[0081] Depend on Figure 4 It can be seen that treatment with 10μg / mL and 20μg / mL white peony extract for 2h can significantly increase the viability of PC12 cells, and the viability of PC12 cells was 79.59±0.87% and 80.70±1.19%, respectively, indicating that white peony extract has a significant in vitro neuroprotective effect.

[0082] 3. Evaluation of the neuroprotective effect of white peony root extract in vivo

[0083] The experimental scheme is as follows: Construct a mouse MCAO model: C57BL / 6 mice were used as subjects. After fasting overnight, the mice were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital, and the right common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) were completely exposed. A thread plug (diameter of 0.22 mm) was inserted into the ICA, advanced to the starting point of the middle cerebral artery (MCA) and left in place for 1.5 hours. After 1.5 hours of occlusion, the thread plug was pulled out for reperfusion and recovered for 24 hours. The sham operation group performed the same surgical operation except for the insertion of the thread plug.

[0084] Experimental grouping and administration: The mice were randomly divided into 4 groups: sham operation group (Sham), model group (MCAO), low-dose white peony extract group (10 mg / kg), and high-dose white peony extract group (20 mg / kg). White peony extract was dissolved in normal saline, and different concentrations of white peony extract were injected intraperitoneally into the high-dose and low-dose groups; the sham operation group and the model group were injected intraperitoneally with the same amount of normal saline. The drugs were administered once a day for 7 consecutive days.

[0085] mNSS score: The mNSS neurological deficit score standard was used to evaluate the neurobehavior of mice that recovered for 24 hours. Scoring criteria: tail lifting test, 1 point for forelimb flexion, 1 point for hindlimb flexion, 1 point for head rotation deviating from the central axis within 30 seconds; 0 point for normal walking, 1 point for inability to walk straight, 2 points for rotation to the paralyzed side, 3 points for falling to the paralyzed side; balance beam test, 0 point for stable balance, 1 point for grasping the edge of the balance beam, 2 points for grasping the balance beam and falling from the balance beam with one foot, 3 points for grasping the balance beam and falling from the balance beam with both feet, or rotating on the balance beam (time>60s), 3 points for trying to keep balance on the balance beam but falling (time>40s), 4 points for trying to keep balance on the balance beam but falling (time>20s), 5 points for not trying to keep balance or grasping the balance beam (time<20s), 6 points for loss of reflexes or abnormal activity, 1 point for not shaking the head when stimulating the external auditory canal, 1 point for not blinking when cotton touches the cornea.

[0086] 2,3,5-Triphenyltetrazolium chloride (TTC) staining: After neurobehavioral assessment, mice were sacrificed and TTC staining was performed to assess the infarct volume. The mouse brain was cut into five coronal sections and incubated in 2% TTC at 37°C in the dark for 30 min. ImageJ software was used to calculate and quantify the infarct volume. The results are shown in Figure 5 .

[0087] Depend on Figure 5It can be seen that continuous administration of white peony extract at a dose of 10 mg / kg and 20 mg / kg for 7 days can significantly reduce the cerebral infarction volume and nerve damage in mice. The cerebral infarction volume was 24.26±2.00% and 16.14±1.26%, respectively, and the mNSS scores of nerve damage were 8.67±1.53 and 6.33±1.00, respectively, indicating that white peony extract has a significant neuroprotective effect in vivo.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for extracting white peony root extract, characterized in that: The following steps are involved: S1. Evenly mix L-proline and glycerol, heat and stir in an oil bath until the mixture is transparent and uniform, to obtain a hydrophilic deep eutectic solvent; S2, mixing betaine and benzyl alcohol evenly, heating and stirring in an oil bath until the mixture is transparent and uniform, to obtain a lipophilic deep eutectic solvent; S3, the hydrophilic deep eutectic solvent obtained in step S1 and the lipophilic deep eutectic solvent obtained in step S2 are uniformly mixed in a certain volume ratio, deionized water is added, and the mixture is heated and stirred in an oil bath to obtain a two-phase deep eutectic solvent system; S4, adding the white peony root powder to the biphasic deep eutectic solvent system obtained in step S3, performing ultrasound-assisted extraction, filtering the filtrate, centrifuging, and discarding the precipitate to obtain a white peony root extract; S5. Load the white peony root extract obtained in step S4 onto an AB-8 macroporous adsorption resin chromatography column, elute with ethanol, collect the eluate, concentrate, and vacuum dry to obtain the white peony root extract.

2. The method for extracting white peony root extract according to claim 1, characterized in that: In step S1, L-proline and glycerol are mixed uniformly in a molar ratio of 1:3, and heated in an oil bath at 80° C. with stirring until the mixture is transparent and uniform, thereby obtaining a hydrophilic low eutectic solvent.

3. The method for extracting white peony root extract according to claim 1, characterized in that: In step S2, betaine and benzyl alcohol are mixed uniformly in a molar ratio of 1:3, and heated in an oil bath at 80° C. with stirring until the mixture is transparent and uniform, thereby obtaining a lipophilic deep eutectic solvent.

4. The method for extracting white peony root extract according to claim 1, characterized in that: In step S3, the hydrophilic deep eutectic solvent obtained in step S1 and the lipophilic deep eutectic solvent obtained in step S2 are uniformly mixed in a volume ratio of 7:3, 50%-60% volume of deionized water is added, and the mixture is heated and stirred in an oil bath at 80° C. for 60 minutes to obtain a two-phase deep eutectic solvent system.

5. The method for extracting white peony root extract according to claim 1, characterized in that: In step S4, the ultrasonic power is 200-250W, the extraction temperature is 50-60°C, the extraction time is 25-30min, the centrifugal speed is 5000-10000r / min, and the centrifugal time is 50-60min.

6. The method for extracting white peony root extract according to claim 1, characterized in that: In step S5, the white peony root extract obtained in step S4 is loaded onto an AB-8 macroporous adsorption resin chromatography column and eluted with ethanol. The loading volume is 0.1-0.2 BV, the elution flow rate is 1.5 BV / h, and each gradient elution is 3-5 BV.

7. The method for extracting white peony root extract according to claim 1, characterized in that: In step S5, the vacuum drying temperature is 50-60°C, the drying time is 100-120min, and the vacuum degree is 60-80Pa.

8. The white peony root extract obtained by the extraction method according to any one of claims 1 to 7.

9. Use of the white peony root extract obtained by the extraction method according to any one of claims 1 to 7 or the white peony root extract according to claim 8 in the preparation of a drug for protecting neurons.

Images