Preparation method and application of gardenia extract

Abstract

The invention provides a preparation method and application of a gardenia extract. Comprising the following steps: 1, extracting; step 2, purification; the invention also relates to application of the gardenia 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 gardenia crocin components, the conditions for purifying the gardenia crocin components are determined, and theoretical basis and technical support are provided for preparing the high-purity gardenia crocin components and realizing industrial production. The method is based on traditional medication experience, is combined with scientific technologies such as activity tracking, separation and purification and the like, and is a strategy of accurately positioning and enriching a core functional substance group, and is a successful example of modern research of traditional Chinese medicines; the concept of synergistic effect of multiple components of the traditional Chinese medicine is reserved, the effectiveness and controllability of the medicine are greatly improved through enrichment, and a foundation is laid for preparing novel medicines for improving and / or 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 of gardenia extract and application thereof. BACKGROUND

[0002] Depression is a common mental disorder characterized by low mood as the main feature. In recent years, with the increase of social psychological pressure, the incidence rate has shown a rising trend year by year.

[0003] Traditional antidepressants (such as SSRIs) have a 30%-50% treatment resistance rate, and are accompanied by side effects such as sexual dysfunction, weight gain, and withdrawal syndrome, so it is urgent to develop safer and more effective alternative therapies. In the search for safer and more comprehensive treatment strategies, traditional Chinese medicine is increasingly valued for its unique advantages: traditional Chinese medicine treatment of depression has the core advantages of multi-target synergistic and overall regulation. Traditional Chinese medicine compounds or single herbs contain multiple active ingredients, which can simultaneously act on neurotransmitter systems (such as 5-HT, NE, DA), hypothalamic-pituitary-adrenal (HPA) axis, neural inflammation, neurotrophic factors (such as BDNF), oxidative stress, and other multiple links closely related to the pathophysiology of depression, achieving "overall regulation, multi-target intervention", which is more in line with the multi-factor pathogenic characteristics of depression; compared with some western medicines (such as SSRIs) that may cause sexual dysfunction, gastrointestinal discomfort, insomnia or sleepiness, withdrawal reactions, etc., the side effects of traditional Chinese medicine (especially compounds) are usually milder, the efficacy is stable, the side effects are relatively few, the patient's tolerance and compliance are better, and long-term use is relatively safe; traditional Chinese medicine can not only improve the core symptoms such as low mood and loss of interest, but also effectively relieve the accompanying somatic symptoms (such as insomnia, loss of appetite, fatigue, pain, etc.), achieve physical and mental treatment, and improve the overall quality of life of patients; some traditional Chinese medicines have advantages in stabilizing mood and regulating constitution, which may help to reduce the risk of recurrence of depression.

[0004] Gardenia is a commonly used traditional Chinese medicine for clearing heat and purging fire, and resolving toxins. Its fruits are widely used to treat "heartburn and anxiety" (i.e. restlessness, similar to anxiety and depression). Modern pharmacological studies have confirmed the significant antidepressant activity of gardenia, and its core active ingredients mainly include two categories of iridoid glycosides and crocin.

[0005] Iridoid glycosides can significantly increase the levels of monoamine neurotransmitters such as serotonin (5-HT), norepinephrine (NE), dopamine (DA) and their metabolites in the hippocampus and prefrontal cortex, regulate neurotransmitters; inhibit the overactivation of the HPA axis caused by chronic stress, reduce the level of plasma corticosterone (CORT), reduce the damage of hippocampal neurons caused by stress, regulate the HPA axis; inhibit neuroinflammatory responses (such as reducing pro-inflammatory factors such as IL-1β, IL-6, TNF-α), reduce oxidative stress damage, promote the expression of neurotrophic factors (such as BDNF), protect neurons from damage, promote neural plasticity, and play a role in neuroprotection and anti-inflammatory; studies have shown that gardenoside and its metabolites may indirectly affect central nervous function by regulating the composition of intestinal flora and its metabolites (such as short-chain fatty acids), participate in the antidepressant effect, and regulate the gut-brain axis.

[0006] Crocins are strong natural antioxidants that can effectively scavenge free radicals (ROS), increase the activity of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), reduce the damage of oxidative stress to neurons, and inhibit NLRP3 inflammasome and other pathways to reduce neuroinflammation; can regulate monoamine neurotransmitters and increase the content of 5-HT, NE, DA and other neurotransmitters in the brain; promote BDNF expression, protect hippocampal neurons from stress-induced damage and apoptosis, improve synaptic plasticity and neurogenesis, and play a neuroprotective role; may exert neuroprotective and antidepressant effects by regulating the glutamatergic system (regulating NMDA receptors); studies have shown that it may regulate monoamine oxidase (MAO, inhibit MAO activity) and reduce the degradation of neurotransmitters.

[0007] The current extraction and purification process of the antidepressant active ingredients of Gardenia jasminoides Ellis has the following technical problems:

[0008] (1) The existing technology generally uses single solvent (water or low concentration ethanol) extraction → concentration → drying process, and the content of crocin in the obtained extract is low, and there are a large amount of polysaccharides (≥30%) and other invalid / interfering components, animal experiments show that its antidepressant activity is weak, and it cannot reach the equivalent dose of chemical drugs, the active ingredients of the crude extract are unclear, and the curative effect is unstable.

[0009] (2) Most of the purification processes of Gardenia jasminoides Ellis use silica gel column chromatography → organic solvent gradient elution, silica gel is used once, which is costly; toxic organic solvents such as chloroform and methanol are commonly used, which can cause solvent residues; the silica gel column has a small processing capacity (column load < 0.1 g of crude drug / g of silica gel), and the separation and purification technology is inefficient and difficult to industrialize.

[0010] (3) The traditional Chinese medicine extraction and separation technology commonly used for Gardenia jasminoides Ellis has a serious waste of energy, and the concentration and drying temperature is > 80℃, which not only causes energy waste, but also leads to the degradation of heat-sensitive crocin I.

[0011] Based on the deficiencies of the above-mentioned existing process, how to prepare high-purity gardenia crocin components by macroporous resin adsorption method has great significance. SUMMARY

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

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

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

[0015] Step 1, extraction

[0016] (1.1) Gardenia medicinal materials are weighed, crushed, and the coarse powder is collected;

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

[0018] (1.3) The alcohol extract is diluted with water to obtain a sample solution;

[0019] Step 2, purification

[0020] The sample solution is loaded, impurities are removed with 30% ethanol solution, the 50% ethanol solution elution part is collected, concentrated, and spray dried to obtain the gardenia extract.

[0021] Preferably, in step 1, the extraction is one of decoction extraction, reflux extraction, immersion extraction, ultrasonic extraction, percolation extraction and microwave extraction.

[0022] Preferably, in step 1, the solvent used in the extraction is one or several of water, ethanol and propanol.

[0023] Preferably, in step 1, the extraction time is 1-5h, and the extraction frequency is 1-3 times.

[0024] Preferably, in step 1, the extraction time is 1h, and the extraction frequency is 2 times.

[0025] Preferably, in step (1.2), the concentration of the ethanol is 60%, and the double amount of the ethanol is 8.

[0026] Preferably, in step 2, the purification comprises: loading----washing----collecting----washing; 30% ethanol is used for washing 4 column volumes; 50% ethanol is used for collecting 4 column volumes; and 100% ethanol is used for washing 4 column volumes.

[0027] Preferably, the purification specifically comprises: D-101 macroporous adsorption resin column separation, wet loading, ethanol gradient elution, 30% ethanol elution 4 column volumes; 50% ethanol elution 4 column volumes; 100% ethanol elution 4 column volumes, spray drying, to obtain gardenia extract.

[0028] The present application also relates to a use of the gardenia extract in the preparation of a medicine for improving and / or treating depression, the gardenia extract being prepared by the above method.

[0029] The present application provides a preparation method and application of a traditional Chinese medicine extract, characterized in that the traditional Chinese medicine extract enriches a type of components with crocin I as an index component in traditional Chinese medicine gardenia as an effective part for treating depression.

[0030] Preferably, the purification process comprises a type of components with crocin I as an index component in traditional Chinese medicine gardenia as an effective part.

[0031] The dosage form of the pharmaceutical composition can be a solid preparation, a liquid preparation or a semi-solid preparation, preferably a tablet, a capsule or an injection, including sustained-release tablets, sustained-release capsules and sustained-release injections.

[0032] The present application has the following advantages:

[0033] (1) In the present application, the content of crocin I in the gardenia crude extract obtained by the water extraction / alcohol extraction method in the prior art is generally <5%, and is mixed with interfering components (gardenoside, polysaccharides, etc.), resulting in dilution of the drug efficacy; in the present application, 30% ethanol is used for impurity removal, water-soluble components are removed, 50% ethanol is used for elution, and the elution solvent is accurately matched based on the polarity characteristics of crocin, so that the content of crocin I in the final product is ≥10% (HPLC detection), which is also significantly higher than the content of crocin I in the crude extract.

[0034] (2) In the present application, 30% ethanol is used for impurity removal, and 50% ethanol is used for elution, so that a type of components with crocin I as an index component are enriched on the resin, achieving the purpose of synergistic effect.

[0035] (3) The present application proves through animal experiments, pharmacological experiments, etc. that the treatment effect of the effective part obtained based on traditional Chinese medicine gardenia prepared by the method of the present application is better than that of crocin I monomer.

[0036] (4) The present application specifies the preparation method of the effective part of gardenia with crocin I as an index, and conducts pharmacodynamic study, laying a foundation for obtaining a pharmaceutical composition with excellent efficacy, clear components and no obvious toxic and side effects;

[0037] (5) The method is based on the traditional medicine experience (gardenia anti-depression), combined with active tracking, separation and purification technology, and the strategy of accurately positioning and enriching the core effective material group, which is a successful example of modernization of traditional Chinese medicine. It not only retains the concept of multi-component synergistic action of traditional Chinese medicine, but also greatly improves the effectiveness and controllability of the drug through enrichment, thereby laying a foundation for preparing new drugs for improving and / or treating depression and anxiety.

[0038] (6) The application establishes the conditions for purifying gardenia crocin components according to the adsorption and analysis characteristics of macroporous adsorption resin on gardenia crocin components, and provides a theoretical basis and technical support for preparing high-purity gardenia crocin components and realizing industrialized production. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 is a flow chart of the preparation method of the gardenia extract according to the present application;

[0040] Figure 2 is a standard curve of crocin I;

[0041] Figure 3 is a graph showing the effect of gardenia extract on the depressive behavior of CUMS mice;

[0042] Figure 4 is a graph showing the effect of gardenia extract on the contents of 5-HT, DA and NE in the hippocampus of CUMS depressive mice. DETAILED DESCRIPTION

[0043] The present application will be described in detail below with reference to specific examples. It should be noted that the following examples are only further illustrations of the present application, and the scope of protection of the present application is not limited to the following examples.

[0044] Example 1

[0045] This embodiment relates to a preparation method of gardenia extract, as shown in Figure 1 , comprising the following steps:

[0046] Step 1, extraction

[0047] (1.1) Weigh the gardenia medicinal material, crush it, and collect the coarse powder;

[0048] (1.2) Double the amount of ethanol, reflux extraction, combine the medicinal liquid, and obtain the alcohol extract;

[0049] (1.3) Dilute the alcohol extract with water to obtain a sample solution;

[0050] Optimization of gardenia extraction process

[0051] (1) Gardenia extraction process orthogonal experiment method and results

[0052] The extraction of Gardeniae Fructus was carried out by heating reflux method. According to the pre-test results, physicochemical properties of medicinal materials and literature reports, ethanol concentration, extraction time, extraction times and solvent volume were used as the investigation factors, three levels were selected for each factor, the content of crocin I in Gardeniae Fructus was used as the investigation index, L9(34) orthogonal table was used to investigate each factor, and the specific factor level arrangement was shown in Table 1 and Table 2.

[0053] Table 1

[0054]

[0055] Table 2

[0056]

[0057]

[0058] Orthogonal test method

[0059] Gardeniae Fructus crude powder was divided into 27 parts, each part was 100.0 g, L9(34) orthogonal experiment was designed according to Table 7, and the filtrate was concentrated under reduced pressure to extract. The content of crocin I was used as the index to optimize the best alcohol extraction process, and the results were shown in Table 3 and Table 4.

[0060] Table 3

[0061]

[0062] Table 4

[0063] Source of variance Sum of squares of deviation Degrees of freedom Mean square F P A 35.34 2 17.67 2915.07 <0.05 B 1.59 2 0.79 131.72 <0.05 C 0.88 2 0.44 72.83 <0.05 D 0.86 2 0.43 71.35 <0.05 Error 0.019590555 18 0.001088364

[0064] The results showed that: taking the content of crocin I as the investigation index, the influence degree of four factors on 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 crocin I, and the best process was determined as A2B1C2D1 according to the actual production situation.

[0065] Verification test

[0066] Gardeniae Fructus crude powder 300 g was divided into 3 parts, each part was 100 g, the best extraction condition was used, that is, 8 times of 60% ethanol was used for reflux extraction for 2 times, each time for 1 h, the filtrate was concentrated under reduced pressure to extract, three groups of parallel experiments were carried out, and the best extraction process was verified. The results were shown in Table 5.

[0067] Table 5

[0068]

[0069] The results show that the content of crocin I is higher than the orthogonal experiment level, the content of effective component is high, the condition is stable, and the process optimization design is reasonable.

[0070] (2) Determination of the content of crocin I in gardenia

[0071] (2.1) Chromatographic conditions and system suitability test

[0072] The chromatographic column was Agilent 5TC-C18 column (4.6 mm x 200 mm, 5 μm) with octadecylsilane-bonded silica gel as the stationary phase; the isocratic elution was carried out with methanol (B)-water (C) (48:52); the detection wavelength was 440 nm; the flow rate was 1.0 mL / min; the column temperature was 25 °C; and the injection volume was 10 μL. The preparation of crocin I reference substance solution was as follows: 25.6 mg of crocin I standard was taken in a 10 mL volumetric flask, which was diluted to the mark with mobile phase (48% methanol) to obtain 256 μg / mL standard solution. The solution was diluted to 128, 64, 32, 16, 8, 4 μg / mL, respectively, to the mark, and then shaken to obtain the solution.

[0073] (2.2) Preparation of test solution

[0074] 0.1 g of gardenia extract sample was accurately weighed and dissolved in 60% methanol, and then transferred to a 25 mL volumetric flask, which was shaken and diluted to the mark with 60% methanol, and then filtered with a 0.45 μm microporous filter

[0075] (2.3) Linearity test

[0076] The standard solution was prepared by mixing 4, 8, 16, 32, 64, and 128 μg / mL of the reference substance. The standard curve was y=74214x-66996, r=0.9997, indicating that the peak area of crocin I showed a good linear relationship in the concentration range of 4-128 μg / mL. See Table 6 and Figure 2 .

[0077] Table 6

[0078]

[0079] (2.4) Precision test The standard solution (16 μg / mL) was injected continuously for 6 times under the above chromatographic conditions, and the average peak area of crocin I was 355591, and the RSD was 1.17%. The results are shown in Table 7, which shows that the instrument precision is good.

[0080] Table 7

[0081] Number of injections Peak area 1 1123297 2 1119416 3 1117307 4 1121236 5 1157341 6 1119236 Average peak area 1126305 RSD (%) 1.17

[0082] (2.5) Stability test The standard solution (16 μg / mL) was determined under the above chromatographic conditions at 0, 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 12.0 h, and the RSD was 0.96%, indicating that Crocin I had good intra-day stability. The results are shown in Table 8.

[0083] Table 8

[0084]

[0085] Step 2, purification

[0086] The sample solution was loaded, impurities were removed with 30% ethanol solution, the elution fraction of 50% ethanol solution was collected, concentrated, and spray dried to obtain the gardenia extract.

[0087] Optimization of gardenia macroporous adsorption resin separation process

[0088] (1) D-101 macroporous adsorption resin pretreatment

[0089] The D-101 macroporous adsorption resin was soaked overnight with 95% ethanol to swell, and the swelled resin was washed with 95% ethanol for about 5 times the column volume until the effluent was clear in a 1:5 mixture of ethanol and distilled water. The resin was washed with distilled water for 5 times the column volume until the effluent was neutral. The resin was activated with 2% hydrochloric acid for about 3 times the column volume and soaked for 2 h. The resin was activated with 2% sodium hydroxide for about 3 times the column volume and soaked for 2 h. The resin was washed with water until neutral, and stored in ethanol. Before loading, the solvent was replaced with water.

[0090] (2) Sample preparation

[0091] A certain amount of gardenia powder (100 g, 300 g, 600 g, 900 g, 1200 g, 1800 g) was weighed, 8 times the amount of 60% ethanol was added, and refluxed for 1 h. The extraction was repeated twice, and the extract was combined. After cooling to room temperature (25°C), the filtrate was collected and concentrated. Water was added for dilution, and the sample was loaded.

[0092] (3) Separation process

[0093] The column separation process was as follows: loading ---- impurity removal ---- sample collection ---- impurity removal; 30% ethanol was used for impurity removal for 4 times the column volume; 50% ethanol was used for sample collection for 4 times the column volume; 100% ethanol was used for impurity removal for 4 times the column volume.

[0094] (4) Analysis of experimental results

[0095] The content of Crocin I in the gardenia extract was analyzed according to the chromatographic conditions and standard curve equation in the determination of the content of Crocin I in gardenia, as shown in Table 9.

[0096] Table 9

[0097]

[0098] Stability of Crocin I in Gardenia Extract

[0099] Through literature research and experimental study, it was found that crocin I is a compound that is easy to hydrolyze. Therefore, the stability of crocin I was investigated in the process optimization research. The stability of crocin I in water solution, 40% ethanol solution and solid powder was investigated at 25°C, 40°C, 60°C, 80°C and 100°C, respectively. The results are shown in Table 10.

[0100] Table 10

[0101]

[0102] The results show that the dry powder of crocin I has good stability, and the stability in 45% ethanol solution is also good. The higher the temperature in water solution, the easier crocin I is to decompose.

[0103] From the above experimental results, the best preparation process of gardenia extract can be obtained

[0104] The gardenia was crushed, and the coarse powder was sieved. 8 times the amount of 60% ethanol was added for reflux extraction for 2 times, 1 h each time. The gardenia extract was obtained by filtration, and was placed at room temperature (25°C). The ethanol was recovered by concentration under reduced pressure at 60°C. The concentrated solution was diluted with water. The filtrate was separated by D-101 macroporous adsorption resin column. The wet sample was loaded, and gradient elution was performed with ethanol. 30% ethanol was used to wash the impurities for 4 column volumes. 50% ethanol was used to collect the sample for 4 column volumes. 100% ethanol was used to wash the impurities for 4 column volumes. Spray drying was performed to obtain the gardenia extract (50% ethanol fraction).

[0105] Example 2

[0106] This example relates to the pharmacodynamic evaluation of gardenia extract fraction

[0107] I. Model establishment

[0108] The animals were 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, i.e. lack of pleasure, which is manifested as reduced motor activity, lack of interest in pleasure, reduced exploration behavior, easy to despair, etc. It has high effectiveness and long duration.

[0109] The experimental animals (C57BL / C mice, body weight 16-18 g) are stimulated by the methods of 1 min tail clamping, 24 h water deprivation, 24 h food deprivation, 5 min 45°C hot water swimming, 24 h light-dark reversal, 1 h restraint, and 5 min 4°C ice water swimming. 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 light-dark reversal, 4 times of 5 min 4°C ice water swimming, 4 times of 5 min 45°C hot water swimming, 4 times of 1 min tail clamping, and 4 times of 1 h restraint.

[0110] The operation method of each stimulus is as follows:

[0111] ① Food deprivation: 24 h of food deprivation, i.e. no feeding from 8:00 am to 8:00 am the next day.

[0112] ② Water deprivation: 24 h of water deprivation, i.e. no water feeding from 8:00 am to 8:00 am the next day.

[0113] ③ Light-dark reversal: At 8:00 am, the animals are placed in a light-dark reversal box without light to make the animals in the dark state; at 20:00, the light of the light-dark reversal box is turned on to make the animals in the light state until 8:00 am the next day.

[0114] ④ Ice water swimming: The animals are placed in a bucket containing 4°C ice water (water depth 15 cm), the mouse's hind foot tips can just touch the bottom of the bucket, and the animals are taken out after 5 min and placed back in the cage.

[0115] ⑤ Hot water swimming: The animals are placed in a bucket containing 45°C hot water (water depth 15 cm), the mouse's hind foot tips can just touch the bottom of the bucket, and the animals are taken out after 5 min and placed back in the cage.

[0116] ⑥ Tail clamping: The mice are placed in a fixation cage, the tail is exposed, and a hemostat is clamped 1 cm from the tail root (the force should not be too large, so that the mouse can emit a cry), and the mouse is pulled out and placed back in the cage after 1 min.

[0117] ⑦ Restraint: The mice are placed in a 10 cm diameter cylinder for 1 h.

[0118] II. Animal grouping and drug administration

[0119] C57BL / 6J mice were randomly divided into groups (9 in each group), namely normal control group (N), model group (M), chronic unpredictable mild stress model group (CUMS), gardenia extract high-dose group (ZZ-H), gardenia extract medium-dose group (ZZ-M), gardenia extract low-dose group (ZZ-L), fluoxetine group (Flu), crocin I group (Cro). After one week of adaptive feeding, the animals could freely eat and drink. The feeding environment temperature was 22±2℃, and the humidity was 45±2%. From 9:00 to 10:00 every day, the animals were respectively given intragastrical administration (C group and N group were given the same volume of normal saline), and the intragastrical administration was continuously performed for 3 weeks.

[0120] III. Behavioral evaluation

[0121] ① Sugar water preference test

[0122] The animals were deprived of water and food for 24 hours before the test. The amount of 1% sucrose solution consumed by the animals in 4 hours was measured. The calculation formula of sugar water preference was: sugar water consumption / total liquid consumption x 100%.

[0123] ② Forced swimming test

[0124] The water temperature in the barrel (barrel height 50 cm, diameter 20 cm) was 23-25℃, and the water depth was 35 cm. The animals were forced to swim for 5 minutes, and the immobility time (s) of the animals was recorded. When the animals in water were in a passive floating state and stopped struggling for more than 3 seconds, it was considered that the animals were in an immobile state. The calculation formula of immobility time was: immobility time / total time x 100%.

[0125] ③ Tail suspension test

[0126] The tail of the animal was fixed with adhesive tape at the rear 1 / 3, and was suspended on a support with the head 15 cm away from the table surface. The animal was filmed, and the C57BL / 6J mice used a white background. The filming was stopped after 6 minutes, and the immobility time of the animal in the last 4 minutes was recorded.

[0127] IV. Determination of the contents of serum and brain tissue neurotransmitters of CUMS model mice

[0128] After the behavioral test, the animals were decapitated to take the brain, and blood was taken. The prefrontal cortex and hippocampus were separated and frozen with liquid nitrogen, and were stored at -80℃. The contents of 5-hydroxytryptamine (5-HT), dopamine (DA), and norepinephrine (NE) were determined according to the ELISA instruction (the ELISA kit was purchased from Shanghai Jiwei Biological Technology Co., Ltd.).

[0129] V. Experimental results

[0130] See Figure 3 and Figure 4 ; as Figure 3 is the effect of gardenia extract on the depressive-like behavior of CUMS mice.

[0131] The sugar water preference test (SPT), tail suspension test (TST) and open field test (OFT) were used to evaluate the improvement of the depression-like behavior of mice by Gardenia extract. Figure 3 From left to right are the results of sugar water preference test, tail suspension test and open field test, as shown in Figure 3 A (the leftmost graph) shows that compared with the normal group, the sugar water preference rate of the depression model group of mice was significantly reduced, indicating that long-term stress stimulation led to the loss of pleasure in the model mice. In contrast, the sugar water preference rate of mice in the high-dose Gardenia group (ZZ-H) and the positive drug fluoxetine (Flu) group was significantly increased after administration, and the active ingredient crocin I (Cro) in Gardenia extract also showed a certain effect on improving the sugar water preference of mice. Figure 3 B (the middle graph) is the evaluation result of the tail suspension test. Compared with the normal group, the tail suspension immobility time of the model group of mice was significantly increased (P<0.001); compared with the model group, the tail suspension immobility time of the mice in the high-dose Gardenia group (ZZ-H) was significantly reduced (P<0.001), indicating that drug intervention can alleviate depressive symptoms and despair in mice. Among the various drug groups, the Flu group can significantly reduce the immobility time of depressed mice, and has a significant antidepressant effect. Figure 3 C (the rightmost graph) is the open field test for evaluating the improvement of the depression-like behavior of mice by Gardenia extract. Compared with the normal group, the central area residence time of the depression model mice was significantly reduced, and the exploration behavior was reduced. After four weeks of administration, the mice were tested, and the results showed that compared with the model group, the high, medium and low dose of Gardenia extract and its active ingredient crocin I group could significantly increase the residence time of the central area of the depressed mice (P<0.05), and the high-dose Gardenia extract group (P<0.001) had a significant effect.

[0132] As shown in Figure 4 Figure is the effect of Gardenia extract on the contents of 5-HT, DA and NE in the hippocampus of CUMS depressed mice;

[0133] 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 used in clinical applications, 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 depressed mice and play an antidepressant role, the contents of three monoamine neurotransmitters in the hippocampus of mice were determined. As shown in Figure 4As shown, compared with the normal group, the contents of 5-HT, DA and NE in the hippocampus tissue of the CUMS model group mice were significantly reduced (P<0.001). The decrease of the levels or functions of monoamine neurotransmitters in the brain is the neurochemical basis of the occurrence of depression, and therefore the significant decrease of the contents of 5-HT, DA and NE in the brain tissue indicates that the CUMS mouse model is successfully established. Compared with the model group, the contents of 5-HT, DA and NE in the hippocampus tissue of the gardenia extract high-, medium- and low-dose treatment groups were significantly increased. Among them, the high-dose gardenia extract group and the positive drug group had a significant effect, and the above results show that the gardenia extract can significantly increase the content of monoamine neurotransmitters in the hippocampus tissue of the CUMS depressed mice, and has a better antidepressant effect.

[0134] The various dosage forms related to the pharmaceutical extract of the present application can be prepared according to the technical specifications and requirements in the pharmaceutical field (such as the methods in the pharmacopoeia, textbooks or other prior art) to meet the needs of clinical treatment and / or treatment of depression and anxiety, and suitable various specifications and dosage forms (including solid preparations, liquid preparations and semi-solid preparations) such as tablets, capsules, injections, including sustained-release tablets, sustained-release capsules, sustained-release injections.

[0135] The preparation method of the dosage forms related to the pharmaceutical extract of the present application belongs to the conventional preparation method in the field of pharmacy, and can be realized by the skilled person in the art according to the prior art (such as the pharmacopoeia, textbooks and other technical specifications in the pharmaceutical field). The conventional preparation method of the above dosage forms will not be described again.

[0136] The specific embodiments of the present application are described above. It should be understood that the present application is not limited to the above specific embodiments, 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 present application.

Claims

1. A method for preparing a gardenia extract, characterized by, The method comprises the following steps: Step 1, extraction (1.1) Weigh the gardenia medicinal material, crush, and collect the coarse powder; (1.2) Double the amount of ethanol, reflux extraction, combine the medicinal liquid, and obtain the alcohol extract; (1.3) Dilute the alcohol extract with water to obtain a sample solution; Step 2, purification Load the sample solution, remove impurities with 30% ethanol solution, collect the 50% ethanol elution part, concentrate, and spray dry to obtain the gardenia extract.

2. The method for preparing gardenia extract as described in claim 1, characterized in that, In step 1, the extraction is one of decocting extraction, reflux extraction, immersion extraction, ultrasonic extraction, percolation extraction, and microwave extraction.

3. The method for preparing gardenia extract as described in claim 1, characterized in that, In step 1, the solvent used in the extraction is one or several of water, ethanol, and propanol.

4. The method for preparing gardenia extract as described in claim 1, characterized in that, In step 1, the extraction time is 1-5 h, and the extraction frequency is 1-3 times.

5. The method for preparing gardenia extract as described in claim 4, characterized in that, In step 1, the extraction time is 1 h, and the extraction frequency is 2 times.

6. The method for preparing gardenia extract as described in claim 1, characterized in that, In step (1.2), the concentration of ethanol is 60%, and the double amount of ethanol is 8.

7. The method for preparing gardenia extract as described in claim 1, characterized in that, In step 2, the purification comprises: loading, removing impurities, collecting, and removing impurities; 30% ethanol removes impurities for 4 column volumes; 50% ethanol collects for 4 column volumes; and 100% ethanol removes impurities for 4 column volumes.

8. The method for preparing gardenia extract as described in claim 7, characterized in that, The purification specifically comprises: D-101 macroporous adsorption resin column separation, wet loading, ethanol gradient elution, 30% ethanol removes impurities for 4 column volumes; 50% ethanol collects for 4 column volumes; 100% ethanol removes impurities for 4 column volumes, spray drying, and obtaining the gardenia extract.

9. Use of a Gardenia extract in the preparation of a medicament for improving and / or treating depression, anxiety. The gardenia extract is the gardenia extract prepared according to claim 1.

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