Preparation method and application of spina date seed extract

Abstract

The invention provides a preparation method and application of a spina date seed extract. Comprising the following steps: 1, extracting; and step 2, purification. The invention also relates to application of the spina date seed extract in preparation of drugs for improving and / or treating depression and anxiety. According to the adsorption and analysis characteristics of the macroporous adsorption resin on the magnoflorine components in the spina date seeds, the conditions for purifying the magnoflorine components in the spina date seeds are determined, and theoretical basis and technical support are provided for preparing the magnoflorine components in the spina date seeds and realizing industrial production. According to the method, scientific technologies such as activity tracking, separation and purification and the like are combined on the basis of a traditional method, and a strategy of accurately positioning and enriching a core functional substance group becomes a successful example of modern research of traditional Chinese medicines; the substance obtained by the preparation method not only retains the concept of synergistic effect of multiple components of traditional Chinese medicines, but also greatly improves the effectiveness and controllability of the medicine through enrichment, and lays a foundation for developing and preparing the medicine for improving and treating depression and anxiety disorder.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine extracts; in particular, it relates to a preparation method and application of Zizyphus jujuba Mill. var. spinosa (Bunge) Hu extract. BACKGROUND

[0002] Depression is an emotional disease of central nervous system abnormalities, with mood regulation disorder as the main clinical feature. In recent years, the cumulative number of global depression patients has exceeded 350 million, and the lifetime prevalence rate is 3.4%. According to the statistics of the World Health Organization, by 2030, depression will become the second largest disease in the world.

[0003] At present, the drugs for treating depression include:

[0004] (1) Selective serotonin reuptake inhibitors (SSRIs) representative drugs include fluoxetine (Prozac), sertraline (Zoloft), paroxetine (Sera), etc. They mainly treat depression by inhibiting the reuptake of 5-hydroxytryptamine and increasing the concentration of 5-hydroxytryptamine in the synaptic cleft. However, their side effects are obvious, including nausea, insomnia, and sexual dysfunction.

[0005] (2) Serotonin and norepinephrine reuptake inhibitors (SNRIs) have side effects including nausea, dizziness, elevated blood pressure, and even withdrawal symptoms.

[0006] (3) Tricyclic antidepressants (TCAs) have significant side effects, and excessive use can cause fatal poisoning.

[0007] Zizyphus jujuba Mill. var. spinosa (Bunge) Hu extract is derived from the dried mature seeds of Ziziphus jujuba Mill. var. spinosa (Bunge) Hu, an important anshen medicinal material recorded in traditional Chinese medicine classics. Its core active ingredients mainly include saponins, flavones and flavone carbon glycosides, alkaloids and other components.

[0008] Modern pharmacological studies have confirmed that Zizyphus jujuba Mill. var. spinosa (Bunge) Hu saponins A and B mainly exert an antidepressant effect through a triple mechanism: first, they regulate neurotransmitters by inhibiting the reuptake of 5-HT and NE in the synaptic cleft, directly increasing the concentration of key monoamine neurotransmitters; second, they promote nerve repair by activating the BDNF / TrkB signaling pathway in the brain, significantly increasing the expression of hippocampal brain-derived neurotrophic factor, repairing neuronal damage and improving neural plasticity; third, they inhibit neural inflammation by effectively blocking the overactivation of microglial cells, reducing the release of inflammatory factors such as TNF-α and IL-6, and reducing the damage of inflammation to neurons. The three together constitute a synergistic mechanism of "neurotransmitter balance-nerve regeneration-anti-inflammatory protection".

[0009] The flavonoid component represented by spinosin mainly plays an antidepressant effect by removing free radicals, repairing mitochondrial oxidative damage, blocking the oxidative stress cascade related to depression, enhancing the function of GABA receptors, prolonging the inhibitory effect of nerves to relieve anxiety, inhibiting the overactivation of the HPA axis, reducing the level of plasma corticosterone, and reducing the toxicity of glucocorticoids on hippocampal neurons.

[0010] Magnoflorine can selectively inhibit monoamine oxidase B (MAO-B), reduce the degradation of norepinephrine (NE) and dopamine (DA), thereby increasing the concentration of synaptic cleft neurotransmitters and enhancing the activity of neurotransmitters, relieving the overexcited central nervous system to play a sedative effect and improve insomnia and anxiety symptoms. Magnoflorine in the Suanzaoren-Chuanxiong medicinal pair can act on 5-HT1AR, 5-HTT and NET receptors at the same time; these results suggest that magnoflorine may be an active ingredient for antidepressant effect.

[0011] It is of great significance to purify magnoflorine in Suanzaoren by macroporous resin adsorption method, high-speed counter-current chromatography and the like. SUMMARY

[0012] The application aims to provide a preparation method and application of Suanzaoren extract.

[0013] The application is achieved by the following technical scheme.

[0014] The application relates to a preparation method of Suanzaoren extract, comprising the following steps.

[0015] Step 1, extraction

[0016] (1.1) Suanzaoren medicinal materials are weighed, and oil is pressed and removed to obtain Suanzaoren coarse powder;

[0017] (1.2) double-amount ethanol is added for reflux extraction, and the medicinal liquid is combined to obtain an alcohol extract;

[0018] (1.3) the alcohol extract is concentrated under reduced pressure to the relative density without alcohol smell to obtain a concentrated solution for standby use;

[0019] Step 2, purification

[0020] The concentrated solution is diluted with an appropriate amount of water, loaded, washed with 10% ethanol solution, and the 30% ethanol solution elution part is collected, concentrated and dried to obtain the Suanzaoren extract.

[0021] Preferably, in step (1.2), the extraction is one of decoction extraction, reflux extraction, immersion extraction, ultrasonic extraction, percolation extraction and microwave extraction.

[0022] Preferably, in step (1.2), the solvent used in the extraction is one or more of water, ethanol.

[0023] Preferably, in step (1.2), the extraction time is 1-5 hours, and the extraction is performed 1-3 times.

[0024] Preferably, in step (1.2), the concentration of the ethanol is 60%, and the doubled amount of the ethanol is 10.

[0025] Preferably, in step (1.2), the extraction is performed 3 times, each time for 1.5 hours.

[0026] Preferably, in step (1.3), the relative density is 1.0-1.1, and the temperature is 60°C.

[0027] Preferably, in step 2, the purification comprises: loading----washing----collecting----washing; 10% ethanol washing for 3 column volumes; 30% ethanol collecting for 3 column volumes; 100% ethanol washing for 3 column volumes.

[0028] Preferably, the purification is specifically: D-101 macroporous adsorption resin column separation, wet loading, ethanol gradient elution, 10% ethanol washing for 3 column volumes; 30% ethanol collecting for 3 column volumes; 100% ethanol washing for 3 column volumes, and drying under reduced pressure to obtain the extract of Zizyphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou.

[0029] The present application also relates to the use of the extract of Zizyphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou in the preparation of a drug for improving and / or treating depression and anxiety.

[0030] The extract of Zizyphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chou according to the present application includes various dosage forms: can be prepared into formulations and various specifications and dosage forms (including solid formulations, liquid formulations, and semi-solid formulations) according to the technical specifications and requirements in the pharmaceutical field (for example, methods in the pharmacopoeia, textbooks, or other existing technologies) to meet the needs of clinical treatment and / or treatment of depression and anxiety; such as tablets, capsules, injections, including sustained-release tablets, sustained-release capsules, sustained-release injections.

[0031] The preparation method of the dosage forms of the drug extract according to the present application belongs to the conventional preparation method in the field of pharmacy, and can be prepared by the skilled person in the art according to the existing technology (for example, the pharmacopoeia, textbooks, and other technical specifications in the pharmaceutical field), and the conventional preparation method of the above dosage forms will not be described herein.

[0032] The present application has the following advantages:

[0033] (1) The method of the present application is a preparation method of magnoflorine effective parts in Zizyphi Spinosae Semen, and through pharmacodynamic study, it lays a foundation for obtaining a pharmaceutical composition with excellent efficacy, clear components and no obvious toxic side effects;

[0034] (2) The method of the present application is a successful example of modernization research of traditional Chinese medicine, which is based on traditional methods, combined with active tracking, separation and purification and other scientific technologies, and the strategy of accurately positioning and enriching the core efficacy material group, the prepared material not only retains the concept of multi-component synergistic action of traditional Chinese medicine, but also greatly improves the drug effectiveness and controllability through enrichment, and lays a foundation for developing and preparing drugs for improving and treating depression and anxiety.

[0035] (3) The present application establishes the conditions for purifying magnoflorine components in Zizyphi Spinosae Semen according to the adsorption and analysis characteristics of macroporous adsorption resin for magnoflorine components in Zizyphi Spinosae Semen, which provides a theoretical basis and technical support for preparing magnoflorine components in Zizyphi Spinosae Semen and realizing industrial production.

[0036] (4) The non-toxic reagent is used in the present application, the separation and purification equipment has low threshold, the reagent has high safety, and industrialization is easy to realize. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is a flow chart of the preparation method of Zizyphi Spinosae Semen extract involved in the present application;

[0038] Figure 2 is a magnoflorine standard curve diagram;

[0039] Figure 3 is a diagram of the effect of Zizyphi Spinosae Semen extract on the depressive-like behavior of CUMS mice;

[0040] Figure 4 is a diagram of the effect of Zizyphi Spinosae Semen extract on the contents of 5-HT, DA and NE in the hippocampus tissue of CUMS depressive mice. DETAILED DESCRIPTION

[0041] The present application will be described in detail below in combination with specific embodiments. It should be pointed out that the following implementation examples are only further illustrations of the present application, and the protection scope of the present application is not limited to the following examples.

[0042] Example 1

[0043] This embodiment relates to a preparation method of Zizyphi Spinosae Semen extract, as shown in Figure 1 , comprising the following steps:

[0044] Step 1, extraction

[0045] (1.1) Zizyphi Spinosae Semen medicinal materials are weighed, pressed to remove oil, and Zizyphi Spinosae Semen coarse powder is obtained;

[0046] (1.2) Double amount of ethanol, reflux extraction, combined liquor, alcohol extract;

[0047] (1.3) The alcohol extract was concentrated under reduced pressure to the relative density, to no alcohol, concentrated liquid, ready for use;

[0048] Optimization of extraction process of Zizyphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chiu

[0049] (1) Orthogonal experiment method and results of Zizyphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chiu extraction process

[0050] The extraction of Zizyphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chiu was carried out by heating reflux method. According to the pre-test results, the physicochemical properties of medicinal materials and literature reports, the ethanol concentration, extraction time, extraction times and solvent volume were used as the observation factors, three levels were selected for each factor, the magnolia base content in Zizyphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chiu was used as the observation index, and L9(34) orthogonal table was used to observe each factor. The specific factor level arrangement is shown in Table 1, Table 2, Table 3 and Table 4.

[0051] Table 1

[0052]

[0053] Table 2

[0054] Numbering Ethanol concentration (%) Extraction time (h) Extraction times (times) Solvent fold 1 1(40) 1(1.0) 1(1) 1(8) 2 1(40) 2(1.5) 2(2) 2(10) 3 1(40) 3(2.0) 3(3) 3(12) 4 2(60) 1(1.0) 3(3) 2(10) 5 2(60) 2(1.5) 1(1) 3(12) 6 2(60) 3(2.0) 2(2) 1(8) 7 3(80) 1(1.0) 2(2) 3(12) 8 3(80) 2(1.5) 3(3) 1(8) 9 3(80) 3(2.0) 1(1) 2(10)

[0055] The orthogonal test method was used to take Zizyphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chiu crude powder, which was divided into 27 parts, each part was 50.0 g, and the L9(34) orthogonal experiment was designed according to Table 7 for extraction, filtration, and the filtrate was concentrated under reduced pressure to extract. The magnolia base content was used as the index to optimize the best alcohol extraction process, and the results are shown in Table 3, and the variance analysis results are shown in Table 4.

[0056] Table 3

[0057]

[0058]

[0059] Table 4

[0060] Variance source Sum of squares of deviation Degrees of freedom Mean square F P A 0.16 2.00 0.08 71.56 <0.05 B 0.09 2.00 0.04 40.62 <0.05 C 0.28 2.00 0.14 128.30 <0.05 D 0.03 2.00 0.02 15.90 <0.05 Error 0.01959055 18.0000 0.00108836

[0061] The results showed that: taking the magnolia base content as the observation index, the influence degree of the four factors on the extraction efficiency was C extraction times (times) > A ethanol concentration (%) > B extraction time (h) > D solvent volume; the variance analysis results showed that each factor had significant influence on the extraction of magnolia base I, and the best process was determined as A2B2C3D2 according to the actual production situation.

[0062] Verification test

[0063] Semen Ziziphi Spinosae crude powder 300 g, parallel into 3 parts, each 100 g, the best extraction conditions, namely 10 times the amount of 60% ethanol reflux extraction 3 times, 1.5 h each time, filter, filter concentrate under reduced pressure to extract, three parallel experiments, the best extraction process verification, the results are shown in Table 5.

[0064] Table 5

[0065]

[0066] The results show that the extraction process of magnolia base content is higher than the orthogonal experiment level, the content of effective components is high, the condition is stable, and the process optimization design is reasonable.

[0067] (2) Magnolia base content determination in Semen Ziziphi Spinosae

[0068] (2.1) Chromatographic conditions and system suitability test

[0069] Shimadzu LC-2030C 3D Plus high performance liquid chromatograph; chromatographic column: Agilent 5TC-C18(2) 250x4.6mm; mobile phase: methanol - one thousandth of triethylamine-ice acetic acid aqueous solution (25:75) isocratic elution; flow rate: 1.0 mL / min; column temperature: 30℃; detection wavelength: 238nm; injection volume: 10μL. Magnolia base reference solution was prepared by weighing 4mg of magnolia base standard in a 100mL volumetric flask, and then adding methanol to the mark. Gradually dilute to 40, 32, 24, 20, 16, 12, 8ug / mL to the mark, shake well, and then dilute to 40ug / mL standard solution.

[0070] (2.2) Preparation of test solution

[0071] Each group of extract samples were weighed 0.1g, dissolved in 60% methanol, transferred to 25ml volumetric flask, added 60% methanol to the mark, shake well, and then the supernatant was filtered with 0.45μm microporous membrane, and then the supernatant was filtered with 0.45μm microporous membrane.

[0072] (2.3) Linear relationship investigation

[0073] The standard solution was prepared by configuring a series of reference solutions with concentrations of 8, 12, 16, 20, 24, 32, 40μg / mL. The standard curve was y=29660x+4613.7, r=0.9997, indicating that the peak area of magnolia base showed a good linear relationship in the concentration range of 8-40μg / mL. See Table 6 and Figure 2 .

[0074] Table 6

[0075]

[0076] (2.4) Precision test

[0077] The standard solution (20 μg / mL) was injected for 6 times under the above chromatographic conditions. The average peak area of magnoflorine was 609340 and the RSD was 0.09%. The results were shown in Table 7, which indicated that the precision of the instrument was good.

[0078] Table 7

[0079] Injection times Peak area 1 609532 2 608549 3 609632 4 608731 5 609542 6 610055 Average peak area 609340 RSD (%) 0.09

[0080] (2.5) Stability test

[0081] The standard solution (20 μg / mL) was injected at 0, 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 12.0 h under the above chromatographic conditions. The RSD was 0.06%, which indicated that the intra-day stability of magnoflorine was good. The results were shown in Table 8.

[0082] Table 8

[0083]

[0084] Step 2, purification

[0085] The concentrated solution was diluted with water, and then loaded. The impurities were washed with 10% ethanol solution. The elution fraction of 30% ethanol solution was collected, concentrated, and dried to obtain the extract of Zizyphi Spinosi Semen.

[0086] Optimization test of purification process of Zizyphi Spinosi Semen

[0087] (1) Pretreatment of D-101 macroporous adsorption resin

[0088] The D-101 macroporous adsorption resin was soaked with 95% ethanol overnight to swell. The swelled resin was washed with 95% ethanol for about 5 times of column volume until the effluent was clear. The effluent was mixed with distilled water at a ratio of 1:5. The effluent was washed with distilled water for 5 times of column volume until it was neutral. The resin was activated with 2% hydrochloric acid for about 3 times of column volume and soaked for 2 h. The resin was activated with 2% sodium hydroxide for about 3 times of column volume and soaked for 2 h. The resin was washed with water until it was neutral, and then stored in ethanol.

[0089] (2) Sample preparation

[0090] A certain amount of Zizyphi Spinosi Semen powder (50 g, 100 g, 200 g, 300 g, 400 g) was weighed, and 10 times of 60% ethanol was added. The mixture was refluxed for 1.5 h. The extraction was repeated for 3 times. The extract was collected and concentrated. The concentrated solution was diluted with water and loaded.

[0091] (3) Separation process

[0092] Column separation process is: sample ---- wash ---- sample ---- wash; 10% ethanol wash 3 column volumes; 30% sample 3 column volumes; 100% ethanol wash 3 column volumes;

[0093] (4) Analysis of experimental results

[0094] Under the chromatographic conditions and standard curve equation of determination of Magnolia flower alkaloid content in Semen Ziziphi Spinosae, the Magnolia flower alkaloid content in Semen Ziziphi Spinosae extract was analyzed, as shown in Table 9.

[0095] Table 9

[0096]

[0097] The optimal preparation process of Semen Ziziphi Spinosae extract is as follows: Semen Ziziphi Spinosae is crushed, coarse powder is screened, and 10 times the amount of 60% ethanol is used for reflux extraction for 3 times, 1.5 h each time; filtration is performed, and the Semen Ziziphi Spinosae extract liquid is placed at room temperature (25°C), and ethanol is recovered by concentration under reduced pressure at 60°C; the concentrated liquid is diluted with water; filtration is performed, and D-101 macroporous adsorption resin column purification is performed; wet sample loading is performed, and ethanol gradient elution is performed; 10% ethanol is used for 3 column volumes of washing; 30% ethanol is used for 3 column volumes of sample collection; 100% ethanol is used for 3 column volumes of washing; and drying is performed under reduced pressure to obtain Semen Ziziphi Spinosae extract (30% ethanol part).

[0098] Example 2

[0099] Evaluation of the pharmacodynamic effect of Semen Ziziphi Spinosae extract

[0100] I. Model establishment

[0101] The animal is exposed to a series of repeated unpredictable mild stimuli within a certain period of time, thereby inducing depression-related behaviors. It mainly simulates the core symptoms of human depression, namely lack of pleasure, which is manifested as reduced motor activity, lack of interest in pleasure, reduced exploration behavior, and easy despair, and has high effectiveness and long duration.

[0102] The experimental animals (C57BL / C mice, body weight 16-18 g) are subjected to tail clamping for 1 min, water deprivation for 24 h, food deprivation for 24 h, 45°C hot water swimming for 5 min, 24 h of day-night reversal, restraint for 1 h, and 4°C ice water swimming for 5 min. One of the 7 stimuli is randomly selected each day for 4 weeks. That is, 4 times of 24 h food deprivation, 4 times of 24 h water deprivation, 4 times of day-night reversal, 4 times of 4°C ice water swimming for 5 min, 4 times of 45°C hot water swimming for 5 min, 4 times of 1 min tail clamping, and 4 times of 1 h restraint.

[0103] The operation method of each stimulus is as follows:

[0104] (1) Food deprivation: 24 h of food deprivation, i.e., no feeding from 8:00 am to 8:00 am the next day.

[0105] (2) Water deprivation: 24h water deprivation, i.e. no water from 8:00 am to 8:00 am the next day.

[0106] (3) Reversed light-dark cycle: at 8:00 am, the animals were put into the reversed light-dark cycle box without light to keep the animals in the dark state; at 20:00, the light of the reversed light-dark cycle box was turned on to keep the animals in the light state until the animals were taken out at 8:00 am the next day.

[0107] (4) Ice water swimming: the animals were put into a bucket containing 4℃ ice water (water depth 15 cm), the tip of the mouse's hind foot just could touch the bottom of the bucket, and the animals were taken out after 5 min and put back into the cage.

[0108] (5) Hot water swimming: the animals were put into a bucket containing 45℃ hot water (water depth 15 cm), the tip of the mouse's hind foot just could touch the bottom of the bucket, and the animals were taken out after 5 min and put back into the cage.

[0109] (6) Tail pinch: the mice were put into a fixed cage, the tail was exposed, and a hemostat was used to pinch the tail 1 cm from the tail root (the force should not be too large, so that the mouse could emit a cry), and then the mouse was taken out and put back into the cage after 1 min.

[0110] (7) Restraint: the mice were put into a cylinder with a diameter of 10 cm and restrained for 1 h.

[0111] II. Animal grouping and administration

[0112] The C57BL / 6J mice were randomly divided into groups (9 mice per group), i.e. normal control group (N), model group (M), chronic unpredictable mild stress model group (CUMS), high-dose S. zaylanica extract group (SZR-H), medium-dose S. zaylanica extract group (SZR-M), low-dose S. zaylanica extract group (SZR-L), fluoxetine group (Flu), magnoflorine group (Mag), and the animals were adaptively fed for one week, during which the animals could freely eat and drink. The feeding environment temperature was 22±2℃, and the humidity was 45±2%. From 9:00 am to 10:00 am every day, the animals were administered by gavage (the animals in groups C and N were given the same volume of normal saline), and the administration by gavage was continuously performed for 3 weeks.

[0113] III. Behavioral evaluation

[0114] (1) Sugar water preference test

[0115] The animals were deprived of water and food for 24 h before the test, and the amount of 1% sucrose solution consumed by the animals in 4 h was determined. The calculation formula of sugar water preference was: sugar water consumption / total liquid consumption x 100%.

[0116] (2) Forced swimming test

[0117] The water temperature (23-25℃) of the round barrel (barrel height 50 cm, diameter 20 cm) was 35 cm deep, and the forced swimming was 5 min. The immobile time (s) of the mouse was recorded. The mouse was in a passive floating state in the water, and stopped struggling for more than 3s, then the mouse was considered to be in an immobile state. The calculation formula of the immobile time was: immobile time / total time x 100%.

[0118] (3) Mouse tail suspension test

[0119] The tail of the mouse was fixed with adhesive tape at the back 1 / 3, suspended on the support, the head was 15 cm away from the table surface, and the video was taken. C57BL / 6J mice used white background. After timing for 6 min, stop, record the mouse immobile time for 4 min.

[0120] (4) Determination of the content of serum and brain tissue neurotransmitters of CUMS model mice

[0121] After the behavior, the mouse was decapitated to take the brain, take the blood, separate the prefrontal cortex and hippocampus, and freeze with liquid nitrogen, and store at-80℃. According to the ELISA instruction (ELISA kit purchased from Shanghai Jiwei Biological Technology Co., Ltd.), the contents of 5-hydroxytryptamine (5-HT), dopamine (DA) and norepinephrine (NE) were determined.

[0122] (5) Experimental results

[0123] as shown in Figure 3 and Figure 4 ; Figure 3 is the effect of Suanzaoren extract on the depressive-like behavior of CUMS mice;

[0124] The sucrose preference test (SPT), tail suspension test (TST) and open field test (OFT) were used to evaluate the improvement of Suanzaoren extract on the depressive-like behavior of mice. Figure 3 From left to right are the results of sucrose preference test, tail suspension test and open field test, as shown in Figure 3 A (the leftmost graph), compared with the normal group, the sucrose preference rate of the depressive model group mice was significantly reduced, indicating that long-term stress stimulation led to the loss of pleasure in the model mice. In contrast, the sucrose preference rate of mice in the high-dose Suanzaoren group (SZR-H) and the positive drug fluoxetine (Flu) group was significantly increased after administration, and the active ingredient magnolia flower base (Mag) in Suanzaoren extract also showed a certain effect on improving the sucrose preference of mice. Figure 3B (middle graph) is the result of the tail suspension test evaluation, compared with the normal group, the tail suspension immobility time of the model group mice increased significantly (P<0.001); compared with the model group, the tail suspension immobility time of the high-dose SZR group (SZR-H) and the medium-dose SZR group (SZR-M) was significantly reduced (P<0.001), indicating that drug intervention can alleviate depressive symptoms and despair in mice. In each drug group, Flu and Mag can significantly reduce the immobility time of depressed mice, and have a significant antidepressant effect. Figure 3 C (rightmost graph) is the open field test to evaluate the improvement of the depressive behavior of mice by the extract of Zizyphus jujuba Mill. Compared with the normal group, the central area residence time of the depressive model mice decreased significantly, and the exploration behavior decreased. After four weeks of administration, the mice were tested, and the results showed that compared with the model group, the high-dose, medium-dose and low-dose Zizyphus jujuba Mill. extract and the active ingredient magnoflorine group can significantly increase the residence time of the central area of the depressive mice (P<0.05), and the high-dose Zizyphus jujuba Mill. extract group (P<0.001) has a significant effect.

[0125] wherein, Figure 4 is the effect of Zizyphus jujuba Mill. extract on the contents of 5-HT, DA and NE in the hippocampus of CUMS depressive mice;

[0126] A large amount of clinical and experimental evidence shows that the occurrence of depression is closely related to the decrease in the activity of the signal transduction pathway of neurotransmitters such as 5-HT, DA and NE in the nervous system. Therefore, the current regulation of monoamine neurotransmission at the synaptic level is the basic mechanism of different types of antidepressants clinically applied, including imipramine, fluoxetine, etc. In order to verify whether the active ingredients can regulate the content of 5-HT, NE and DA neurotransmitters in the brain tissue of depressive mice and play an antidepressant effect, the contents of three kinds of monoamine neurotransmitters in the hippocampus of mice were determined. As shown in Figure 4 compared with the normal group, the contents of 5-HT, DA and NE in the hippocampus of the CUMS model group mice decreased significantly (P<0.001). The decrease in the level or function of monoamine neurotransmitters in the brain is the neurochemical basis for the occurrence of depression, so the significant decrease in the contents of 5-HT, DA and NE in the brain tissue indicates that the CUMS mouse model of depression is successfully established. Compared with the model group, the contents of 5-HT, DA and NE in the hippocampus of the mice in the high-dose, medium-dose and low-dose Zizyphus jujuba Mill. extract treatment groups increased significantly. Among them, the high-dose Zizyphus jujuba Mill. extract group and the positive drug group had a significant effect. The above results show that Zizyphus jujuba Mill. extract can significantly increase the content of monoamine neurotransmitters in the hippocampus of CUMS depressive mice, and has a better antidepressant effect.

[0127] The application establishes the conditions for purifying the magnoflorine components of Zizyphus jujuba according to the adsorption and analysis characteristics of the macroporous adsorption resin on the magnoflorine components, and provides a theoretical basis and technical support for preparing the magnoflorine components of Zizyphus jujuba and realizing industrialized production. The method is a successful example of modernization of traditional Chinese medicine, which is based on the traditional method, combined with scientific technologies such as active tracking, separation and purification, and the strategy of accurately positioning and enriching the core effective material group. The prepared material not only retains the concept of multi-component synergistic action of traditional Chinese medicine, but also greatly improves the drug effectiveness and controllability through enrichment, thereby laying a foundation for developing and preparing drugs for improving and treating depression and anxiety.

[0128] The specific embodiments of the application are described above. It should be understood that the application is not limited to the specific embodiments described above, and various modifications or changes can be made by those skilled in the art within the scope of the claims, which do not affect the essence of the application.

Claims

1. A method for preparing a Chinese date seed extract, characterized in that: The following steps are involved: Step 1, Extract (1.1) Weigh the Chinese jujube seeds and press to remove the oil to obtain coarse Chinese jujube seed powder; (1.2) Double the amount of ethanol, reflux extraction, combine the drug solutions, and obtain the alcohol extract; (1.3) Concentrate the alcohol extract under reduced pressure to a relative density and until there is no alcohol smell, to obtain a concentrated solution for later use; Step 2, purification The concentrate was diluted with an appropriate amount of water, loaded onto the sample, washed with 10% ethanol solution, and the eluted portion with 30% ethanol solution was collected, concentrated, and dried to obtain the jujube seed extract.

2. The method for preparing the Chinese date seed extract according to claim 1, wherein In step (1.2), the extraction is one of decoction extraction, reflux extraction, maceration extraction, ultrasonic extraction, percolation extraction, and microwave extraction.

3. The method for preparing the Chinese date seed extract according to claim 1, wherein In step (1.2), the solvent used in the extraction is one or more of water and ethanol.

4. The method for preparing the Chinese date seed extract according to claim 1, wherein In step (1.2), the extraction time is 1 to 5 hours, and the extraction times are 1 to 3 times.

5. The method for preparing the Chinese date seed extract according to claim 1, wherein In step (1.2), the concentration of the ethanol is 60%, and the doubled amount of the ethanol is 10.

6. The method for preparing the Chinese date seed extract according to claim 1, wherein In step (1.2), the extraction was performed 3 times, each time for 1.5 hours; 7. The method for preparing the Chinese date seed extract according to claim 1, characterized in that in step (1.3), the relative density is 1.0-1.1 and the temperature is 60°C.

8. The method for preparing the Chinese date seed extract according to claim 1, wherein In step 2, the purification includes: sample loading - washing - sample collection - washing; wherein, 10% ethanol is used to wash impurities for 3 column volumes; 30% ethanol is used to collect the sample for 3 column volumes; and 100% ethanol is used to wash impurities for 3 column volumes.

9. The method for preparing the Chinese date seed extract according to claim 8, wherein: The purification is specifically as follows: separation with a D-101 macroporous adsorption resin column, wet loading, ethanol gradient elution, washing with 10% ethanol for 3 column volumes; collecting samples with 30% ethanol for 3 column volumes; washing with 100% ethanol for 3 column volumes, and drying under reduced pressure to obtain the jujube seed extract.

10. Use of a jujube seed extract in preparing a drug for improving and / or treating depression and anxiety, characterized in that: The Chinese jujube seed extract is the Chinese jujube seed extract prepared according to claim 1.

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