Application of trillium tschonoskii extract in preparation of medicine for treating depression

Abstract

The invention relates to the technical field of depression treatment, in particular to application of trillium tschonoskii extract in preparation of a medicine for treating depression. The extraction method of the trillium tschonoskii extract comprises the following steps: S1, adding an ethanol solution into trillium tschonoskii according to a solid-liquid ratio of 1: (10-12), and performing reflux extraction at 80-90 DEG C to obtain an extracting solution; s2, performing primary concentration on the extracting solution, and then adding macroporous resin for elution to obtain eluent; performing centrifugation, secondary concentration and freeze-drying on the eluent to obtain a trillium tschonoskii extract; the trillium tschonoskii extract is adopted as the medicine for treating depression, the symptoms of depression and psychotic depression of mice can be obviously relieved, meanwhile, due to the fact that the trillium tschonoskii extract is a natural substance and has no toxic or side effect, the price of the trillium tschonoskii extract is far lower than that of existing western medicine for treating depression, and the trillium tschonoskii extract can be conveniently and widely applied to treatment of depression.

Description

Technical Field

[0001] The present invention relates to the technical field of the treatment of depression, and in particular to the application of Trillium tschonoskii Maxim. extract in the preparation of drugs for treating depression. Background Art

[0002] Depression, also known as depressive disorder, is a mental disorder with a high incidence, a high clinical cure rate, a low treatment acceptance rate, and a high recurrence rate. Clinically, depressive disorders can be classified into mild, moderate, and severe according to the number, type, and severity of symptoms. According to the DSM-5 (Diagnostic and Statistical Manual of Mental Disorders, 5th Edition), the diagnostic criteria for depression are as follows: A. During the same 2-week period, 5 or more of the following symptoms occur, representing a change compared to previous functioning (where at least 1 item is (1) depressed mood or (2) loss of interest or pleasure): 1. Depressed mood almost every day and for most of the day, which can be a subjective report (e.g., feeling sad, empty, hopeless) or observed by others (e.g., manifested as crying).

[0003] 2. Markedly diminished interest or pleasure in all, or almost all, activities almost every day and for most of the day (either a subjective statement or an observed change).

[0004] 3. Significant weight loss when not dieting, or weight gain (e.g., a weight change of more than 5% of the original weight in a month), or decreased or increased appetite almost every day (note: in children, it may be manifested as failure to reach the standard weight).

[0005] 4. Insomnia or hypersomnia almost every day.

[0006] 5. Psychomotor agitation or retardation almost every day (observable by others, not just a subjective feeling of restlessness or sluggishness).

[0007] 6. Fatigue or loss of energy almost every day.

[0008] 7. Feelings of worthlessness or excessive, inappropriate guilt almost every day (which can reach delusional levels and is not just self-reproach or guilt due to illness).

[0009] 8. Diminished ability to think or concentrate, or indecisiveness almost every day (either a subjective statement or an observed change).

[0010] 9. Recurrent thoughts of death (not just fear of death), recurrent suicidal ideation without a specific plan, or a suicide attempt, or a specific plan for committing suicide.

[0011] B. These symptoms cause clinically significant distress or impairment in social, occupational, or other important areas of functioning.

[0012] C. These symptoms are not attributable to the physiological effects of a substance or another medical condition.

[0013] Meeting 5 or more of the criteria in Diagnostic Criterion A is considered major depressive disorder.

[0014] Meeting Diagnostic Criteria A + B + C simultaneously constitutes a major depressive episode.

[0015] Antidepressants can be classified into three categories according to their mechanism of action and the time of their introduction: first-generation antidepressants, which mainly include non-selective monoamine oxidase inhibitors and tricyclic antidepressants, with representative drugs such as phenelzine; second-generation antidepressants, which mainly include reversible and selective monoamine oxidase inhibitors and tetracyclic antidepressants, with representative drugs such as moclobemide; third-generation antidepressants, mainly selective serotonin reuptake inhibitors, selective norepinephrine reuptake inhibitors, and dual reuptake inhibitors of these two neurotransmitters, with representative drugs such as fluoxetine and milnacipran. Currently, drugs based on this mechanism are relatively more commonly used in clinical practice. In addition to drug treatment, physical therapy is also one of the commonly used means for the treatment of depression, such as electroconvulsive therapy, sleep deprivation, and psychotherapy.

[0016] First-generation antidepressants have more adverse reactions and have withdrawn from first-line clinical use; currently, second-generation and third-generation antidepressants are more commonly used in clinical practice. However, for the current drug-based treatment methods, due to the occurrence of adverse reactions, patient compliance is poor. For moderate and severe depression patients, the effective rate of drug treatment is not high, and patients are prone to recurrence. Even some patients have obvious central nervous system side effects during drug treatment. And physical therapy also has certain drawbacks; for example, electroconvulsive therapy is mainly used for patients with poor drug compliance. The patient needs to be anesthetized in advance and then undergo electrostimulation treatment, which has a certain risk of anesthesia death. At the same time, electroconvulsive therapy will also seriously damage the cognitive function of depressive patients. SUMMARY OF THE INVENTION

[0017] The object of the present invention is to provide the application of Trillium tschonoskii Maxim. extract in the preparation of drugs for the treatment of depression.

[0018] To achieve the above object, the present invention adopts the following technical solutions: The application of Trillium tschonoskii Maxim. extract in the preparation of drugs for the treatment of depression, wherein the extraction method of the Trillium tschonoskii Maxim. extract comprises the following steps: S1. Add an ethanol solution to Trillium tschonoskii Maxim. at a material-liquid ratio of 1:10 - 12, and reflux and extract at 80 - 90 °C to obtain an extract. S2. Concentrate the extract once, then add it to macroporous resin for elution to obtain an eluate; during the elution process, first use an ethanol aqueous solution with a mass concentration of 10 - 20% and a volume of 5 - 10 BV for elution at an elution rate of 2 - 3 BV / h; then use an ethanol aqueous solution with a mass concentration of 70 - 80% and a volume of 5 - 10 BV for elution at an elution rate of 2 - 3 BV / h, and collect the eluate of the ethanol aqueous solution with a mass concentration of 70 - 80% for subsequent centrifugation; concentrate it twice and freeze-dry to obtain the Trillium tschonoskii Maxim. extract.

[0019] In one preferred embodiment, the material-liquid ratio is in the form of the mass of the raw material: the volume of the solvent. For example, a material-liquid ratio of 1:10 g / mL means that 1 part by mass of the raw material corresponds to 10 parts by volume of the solvent.

[0020] In one preferred embodiment, the ethanol solution is an ethanol aqueous solution with a mass concentration of 40% - 60%.

[0021] In one preferred embodiment, the number of times of reflux extraction is 2 - 3 times.

[0022] In one preferred embodiment, the first concentration is to concentrate the extract to a concentration 3 - 5 times the original.

[0023] In one preferred embodiment, the macroporous resin is a styrene-type non-polar copolymer macroporous resin.

[0024] In one preferred embodiment, the macroporous resin is HPD-100 macroporous resin, and the addition amount is 100 - 300 g.

[0025] In one preferred embodiment, during the elution process, the height ratio of the sample layer to the resin layer is 1 - 2:1 - 2.

[0026] In one preferred embodiment, during the elution process, first use an ethanol aqueous solution with a mass concentration of 10 - 20% and a volume of 5 - 10 BV for elution at an elution rate of 2 - 3 BV / h; then use an ethanol aqueous solution with a mass concentration of 70 - 80% and a volume of 5 - 10 BV for elution at an elution rate of 2 - 3 BV / h, and collect this part of the eluate for subsequent centrifugation; The eluate obtained by eluting with an ethanol aqueous solution with a mass concentration of 10 - 20% is impurities and is discarded.

[0027] In one preferred embodiment, the rotation speed of centrifugation is 3000 - 4000 r / min, and the time is 10 - 15 min.

[0028] In one preferred embodiment, the secondary concentration is to concentrate the extract to a concentration 3-5 times the original.

[0029] In one preferred embodiment, the freeze-drying process is as follows: the cold trap temperature is -47 to -48 degrees, and the vacuum degree is 13 to 15 Pa.

[0030] In one preferred embodiment, the drug further comprises other active ingredients for treating depression.

[0031] In one preferred embodiment, the drug further comprises other pharmaceutically acceptable excipients.

[0032] In one preferred embodiment, the dosage form of the drug is decoction, pill, capsule, tablet, powder or granule.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows: In the present invention, using Trillium tschonoskii Maxim. extract as a drug for treating depression can significantly relieve the depressive symptoms and psychogenic depressive symptoms of mice. At the same time, since the Trillium tschonoskii Maxim. extract is a natural substance, it has no toxic and side effects, and its price is much lower than that of existing western medicines for treating depression, which is convenient for wide application in the treatment of depression. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the appearance characteristics of Trillium tschonoskii Maxim.; Figure 2 is the process flow chart for extracting Trillium tschonoskii Maxim. extract; Figure 3 is the HE staining diagram of the histopathological effect of Trillium tschonoskii Maxim. extract 1 on the hippocampal tissue of mice in Example 1; In the figure: A is the normal control group; B is the model control group; C is the fluoxetine hydrochloride group; D is the low-dose group of Trillium tschonoskii Maxim. extract 1; E is the medium-dose group of Trillium tschonoskii Maxim. extract 1; F is the high-dose group of Trillium tschonoskii Maxim. extract 1, G is the high-dose group of Trillium tschonoskii Maxim. water extract; H is the high-dose group of Trillium tschonoskii Maxim. alcohol extract. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] The present invention is not limited to the following specific embodiments. Those of ordinary skill in the art can implement the present invention in other various specific embodiments according to the content disclosed in the present invention, or those that adopt the design structure and idea of the present invention and make simple changes or modifications all fall within the protection scope of the present invention. It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0036] Example 1

[0037] Method 1: Extraction, enrichment and purification of Trillium tschonoskii Maxim. extract 1 Experimental materials: Trillium tschonoskii Maxim. is a perennial herb of the genus Trillium in the Melanthiaceae family. It is sweet in taste, neutral in nature, and slightly poisonous, and has been included in the "Hubei Chinese Medicinal Materials Standard".

[0038] Trillium tschonoskii Maxim., such as Figure 1 . Origin: Northeast China. Product batch number: 241101. Production date: November 20, 2024 Experimental process: According to Figure 2 the technological process diagram shown, the extraction is carried out as follows: 1. Extraction Take 500 g of Trillium tschonoskii Maxim., according to the solid-liquid ratio of 1:12 g / mL, add an ethanol aqueous solution with a mass concentration of 50%, and reflux and extract twice at a temperature of 80 - 90 °C, 2 h each time.

[0039] 2. Enrichment with HPD-100 (styrene-type non-polar copolymer) macroporous resin After concentrating to 1 / 3 of the original volume, add 200 g of HPD-100 macroporous resin, and spin on a rotary evaporator until it does not stick to the wall and the particles are relatively distinct. Then load the column, and the thickness ratio of the sample layer to the blank resin layer (packing layer) is 1:1. Elute with 5 BV (1 BV = 2.1 L) of an ethanol aqueous solution with a mass concentration of 20% at a flow rate of 3 BV / h. This part is impurities and is discarded. Then elute with 5 BV of an ethanol aqueous solution with a mass concentration of 70% at a flow rate of 3 BV / h, and collect the eluate of this part.

[0040] 3. Concentration treatment Use a rotary evaporator to concentrate the 70% ethanol part, centrifuge at 3500 r / min for t = 10 min to obtain insoluble matter and centrifugate. The centrifugate is continuously concentrated with a rotary evaporator. When the concentration is tripled and the volume is about 200 mL, put the insoluble matter and the concentrated solution into a -80 °C refrigerator for freezing, and then put them into a freeze dryer for freeze-drying. The freeze-drying process is: the cold trap temperature is -47 to -48 °C, and the vacuum degree is 13 to 15 Pa; finally, about 39.67 g of powdery Trillium tschonoskii Maxim. extract 1 is obtained.

[0041] Method 2: Preparation of water extract of Trillium tschonoskii Maxim. Preparation method of water extract of Trillium tschonoskii Maxim.: Take 500 g of Trillium tschonoskii Maxim., according to the solid-liquid ratio of 1:12 g / mL, decoct with water twice, combine the filtrates and concentrate them, and then dry them in an oven to obtain 92.85 g of water extract of Trillium tschonoskii Maxim.

[0042] Method 3: Preparation of ethanol extract of Trillium tschonoskii Maxim. Preparation method of ethanol extract of Trillium tschonoskii Maxim: Weigh 500 g of Trillium tschonoskii Maxim, and according to the material-liquid ratio of 1:12 g / mL, put it into a round-bottomed flask and add 75% ethanol as the extraction solvent. Fix the round-bottomed flask on the heating reflux device and heat it under reflux at 80 °C for 3 h. After extraction, filter, transfer the obtained extract to a rotary evaporator, and concentrate it under reduced pressure. The concentration temperature is generally controlled at 40 - 60 °C. After concentrating to 1 / 3 of the original volume, the concentrated solution of the ethanol extract of Trillium tschonoskii Maxim is obtained. Transfer the concentrated solution to a petri dish and dry it in a low-temperature drying oven to obtain 31.28 g of solid powder of the ethanol extract of Trillium tschonoskii Maxim.

[0043] 1. Effect on reserpine-induced mouse depression model Test sample Trillium tschonoskii Maxim, batch number: 20240109, expiration date: 2022.09, provided by Hunan Prima Pharmaceutical Research Center Co., Ltd.

[0044] Reference substance Clomipramine Hydrochloride Tablets, batch number: LM202203, product of Jiangsu Nhwa Pharmaceutical Co., Ltd.

[0045] Experimental animals 90 SPF-grade ICR mice, weighing 15.2 - 28.8 g, half male and half female, purchased from Changsha Tianqin Biotechnology Co., Ltd., experimental animal production license number: SCXK(Xiang)2019 - 0013; experimental animal quality certificate number: 430726221100110776, raised in area D of the barrier environment laboratory of Hunan Prima Pharmaceutical Research Center Co., Ltd., experimental animal use license number: SYXK(Xiang)2020 - 0015.

[0046] Main reagents Reserpine injection, specification: 1 mL:1 mg, batch number: 2006041, Tianjin KingYork Pharmaceutical Co., Ltd.

[0047] Main instruments AUY220 type analytical balance, AUW-220D type analytical balance, ME2002E / 02 type electronic balance, products of Mettler company.

[0048] Test method Grouping and administration Ninety ICR mice qualified for quarantine, weighing 18.2 - 21.4 g, with half males and half females, were randomly divided into a normal control group, a model control group, a clomipramine hydrochloride tablet group (9.75 mg / kg), a low- and high-dose group of aqueous extract of Trillium tschonoskii Maxim. (70, 280 mg / kg), a low- and high-dose group of ethanol extract of Trillium tschonoskii Maxim. (70, 280 mg / kg), and a low- and high-dose group of Trillium tschonoskii Maxim. extract 1 (70, 280 mg / kg), with 10 mice in each group, half males and half females. The normal control group and the model control group were intragastrically administered with pure water at a volume of 20 mL / kg. The mice in the other groups were intragastrically administered with the corresponding medicinal solutions once a day for 7 consecutive days. One hour after the last administration, except for the mice in the normal control group which were intraperitoneally injected with 0.9% sodium chloride injection, the mice in the other groups were intraperitoneally injected with reserpine at 2 mg / kg, with an injection volume of 20 mL / kg. After injection, the eyelid closure degree and anal temperature were measured.

[0049] Experimental results 1. Effects of different extraction methods of Trillium tschonoskii Maxim. on eyelid closure degree and anal temperature in reserpine-induced depressive model mice Compared with the model control group, the eyelid closure degrees of the mice in the low- and high-dose groups of aqueous extract of Trillium tschonoskii Maxim., the low- and high-dose groups of ethanol extract of Trillium tschonoskii Maxim., and the low- and high-dose groups of Trillium tschonoskii Maxim. extract 1 were significantly decreased ( P ≤0.01), and the anal temperatures were significantly increased ( P ≤0.01). Compared with the low-dose group of Trillium tschonoskii Maxim. extract 1, the eyelid closure degrees of the mice in the low- and high-dose groups of aqueous extract, the low- and high-dose groups of ethanol extract were significantly increased ( P ≤0.05 or P ≤0.01), and the anal temperatures of the mice in the high-dose group of aqueous extract, the low- and high-dose groups of ethanol extract were significantly decreased ( P ≤0.05). Compared with the high-dose group of Trillium tschonoskii Maxim. extract 1, the eyelid closure degrees of the mice in the low- and high-dose groups of aqueous extract, the low- and high-dose groups of ethanol extract were significantly increased ( P ≤0.05 or P ≤0.01), and the anal temperatures were significantly decreased. The phenomenon of eyelid ptosis in the clomipramine hydrochloride tablet group was significantly decreased ( P ≤0.01), and the anal temperature was significantly increased ( P ≤0.01).

[0050]

[0051] Note: Compared with the normal control group, ++ P ≤0.05; compared with the model control group, * P ≤0.05, ** P ≤0.01. Compared with the low-dose group of Trillium tschonoskii Maxim. extract 1 #P ≤0.05, ## P ≤0.01; Compared with the high-dose group of Trillium tschonoskii Maxim. extract 1 ▲ P ≤0.05, ▲▲ P ≤0.01.

[0052] Experimental summary The low and high doses of Trillium tschonoskii Maxim. extract 1 can significantly reduce the eyelid closure degree of mice and significantly increase the anal temperature. The results show that Trillium tschonoskii Maxim. extract 1 has significant antidepressant effects, and its antidepressant effects are significantly higher than those of the aqueous extract and alcohol extract of Trillium tschonoskii Maxim..

[0053] Example 2 Effect of Trillium tschonoskii Maxim. extract 1 on a rat depression model induced by olfactory bulbectomy Test materials Test samples Trillium tschonoskii Maxim. extract 1, batch number: 240201, expiration date: February 15, 2026, extracted from Example 1.

[0054] Reference substances Fluoxetine hydrochloride, batch number: 202410311, specification: 10 mg / tablet, expiration date: October 30, 2024, product of Changzhou Siyao Pharmaceutical Co., Ltd.

[0055] Experimental animals 70 SPF-grade male WKY rats, weighing 151.8 - 218.6 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., production license number of experimental animals: SCXK (Beijing) 2021-0006, quality certificate number of experimental animals: 110011231107853326, raised in Area D of the barrier environment laboratory of Hunan Prima Pharmaceutical Research Center, experimental animal use license number: SYXK (Hunan) 2020-0015.

[0056] Main instruments JLBehv animal behavior video analysis system, product of Shanghai Jiliang Software Technology Co., Ltd.; ALC-H electric digital animal brain stereotaxic apparatus, product of Shanghai Aolte Biotechnology Co., Ltd.; ALC-CED type animal skull drill: product of Shanghai Aolte Biotechnology Co., Ltd.

[0057] Test methods Animal modeling Ninety SPF-grade male WKY rats with a body weight of 151.8 - 218.6 g were selected and randomly divided into a normal control group of 10 rats and a model group of 80 rats according to body weight. Except for the normal control group, after the rats in the model group were induced anesthesia with isoflurane, the rats were fixed on a stereotaxic apparatus for the brain. The skin of the rat's head was disinfected with iodophor and then cut open to expose the three sutures (coronal suture, sagittal suture, lambdoid suture) and two fontanelles (anterior fontanelle, posterior fontanelle). At 8 mm in front of the anterior fontanelle and 2 mm on both sides of the midline, two holes with a diameter of about 2 mm were drilled with an electric drill, and after the olfactory bulbs were destroyed with a special blunt needle, they were aspirated. After hemostasis, packing, bone wax sealing, and anti-infection treatment of the rat's wound, the skin was sutured. Olfactory bulbectomy-induced depression mainly corresponds to major depressive disorder (MDD) clinically.

[0058] Grouping and administration Fourteen days after the operation, the surviving rats were selected and randomly divided into 7 groups according to the sucrose preference index, namely the model control group, the fluoxetine hydrochloride tablet group, the low, medium, and high-dose groups of Trillium tschonoskii Maxim. extract 1, the high-dose group of Trillium tschonoskii Maxim. water extract, and the high-dose group of Trillium tschonoskii Maxim. alcohol extract, with 10 rats in each group. The normal control group and the model control group were intragastrically administered pure water, and the rats in the other groups were intragastrically administered the corresponding medicinal solutions, with a dosing volume of 10 mL / kg, once a day, for 45 consecutive days. After the last dose, relevant behavioral tests were performed.

[0059] Detection indexes 1 Sucrose preference test: The experiment was divided into an adaptation period, a training period, and a test period. During the adaptation period, the animals were given two bottles of 1% sucrose water to adapt to sucrose drinking for 24 h. During the training period, the animals were given one bottle of 1% sucrose water and one bottle of pure water for 24 h. During the test period, the animals were not fasted before the test and were deprived of water for 16 h. Similarly, the animals were given one bottle of 1% sucrose water and one bottle of pure water, and the test time was 24 h. To avoid the influence of position preference, during the test period, the positions of the two bottles were exchanged. After the test, the sucrose preference index was calculated, and the sucrose preference index = sucrose consumption / (sucrose consumption + pure water consumption) × 100%.

[0060] 2 Forced swimming test: The JL Behv forced swimming video analysis system was used to detect the static time and activity time of the rats within 5 min.

[0061] Experimental results 1 Effect of Trillium tschonoskii Maxim. extract 1 on the sucrose preference index of the olfactory bulbectomy rat depression model As shown in Table 2, compared with the normal control group, the sucrose preference index of the rats in the model control group was significantly decreased (P ≤ 0.01). Compared with the model control group, the sucrose preference indexes of the rats in the low, medium, and high-dose groups of Trillium tschonoskii Maxim. extract 1 were significantly increased ( P ≤ 0.05 or P≤0.01). Compared with the high-dose groups of aqueous extract and ethanol extract of Trillium tschonoskii Maxim., the sucrose preference indexes of the middle- and high-dose groups of Trillium tschonoskii Maxim. extract 1 in rats were significantly increased ( P ≤0.05).

[0062]

[0063] Note: Compared with the normal control group, ++ P ≤0.01; compared with the model control group, * P ≤0.05, ** P ≤0.01. Compared with the high-dose groups of aqueous extract and ethanol extract of Trillium tschonoskii Maxim., # P ≤0.05.

[0064] 2 Effects of Trillium tschonoskii Maxim. extract 1 on the immobility time of rats in forced swimming As shown in Table 3, compared with the normal control group, the immobility time of rats in the model control group during forced swimming was significantly increased (P≤0.01). Compared with the model control group, the immobility time of rats in the middle- and high-dose groups of Trillium tschonoskii Maxim. extract 1 during forced swimming was significantly decreased (P≤0.05 or P≤0.01). The immobility time of rats in the fluoxetine hydrochloride tablet group during forced swimming was significantly decreased ( P ≤0.01). Compared with the high-dose groups of aqueous extract and ethanol extract of Trillium tschonoskii Maxim., the immobility time of rats in the high-dose group of Trillium tschonoskii Maxim. extract 1 during forced swimming was significantly decreased ( P ≤0.01). Compared with the high-dose group of ethanol extract of Trillium tschonoskii Maxim., the immobility time of rats in the high-dose group of Trillium tschonoskii Maxim. extract 1 during forced swimming was significantly decreased ( P ≤0.01).

[0065]

[0066] Note: Compared with the normal control group, ++ P ≤0.01; compared with the model control group, * P ≤0.05, ** P ≤0.01. Compared with the high-dose groups of aqueous extract and ethanol extract of Trillium tschonoskii Maxim., ## P ≤0.01.

[0067] Experimental summary Low, middle, and high doses of Trillium tschonoskii Maxim. extract 1. The middle and high doses of Trillium tschonoskii Maxim. extract 1 can significantly reduce the immobility time during forced swimming. Compared with the high-dose group of ethanol extract of Trillium tschonoskii Maxim., the middle and high doses of Trillium tschonoskii Maxim. extract 1 can significantly increase the sucrose preference index of rats. The high dose of Trillium tschonoskii Maxim. extract 1 can significantly reduce the immobility time of rats during forced swimming. The results show that Trillium tschonoskii Maxim. extract 1 has a significant effect on improving the depressive behavior of rats, and its antidepressant effect is better than that of the aqueous extract and ethanol extract of Trillium tschonoskii Maxim.

[0068] Example 3 Effect of Trillium tschonoskii Maxim. extract 1 on a mouse model of depression induced by chronic unpredictable stress Test materials Test substance Trillium tschonoskii Maxim. extract 1, batch number: 240201, expiration date: February 15, 2026, prepared according to the example

[0069] Reference substance Fluoxetine hydrochloride tablets, batch number: 202410311, specification: 10 mg / tablet, expiration date: October 30, 2024, product of Changzhou Siyao Pharmaceutical Co., Ltd.

[0070] Experimental animals 80 SPF-grade male C57 mice, weighing 16.7 - 21.9 g, purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., production license number for experimental animals: SCXK(Xiang)2019 - 0004, quality certificate number for experimental animals: 430727231101501437, housed in area D of the barrier environment laboratory of Hunan Prima Pharmaceutical Research Center Co., Ltd., use license number for experimental animals: SYXK(Xiang)2020 - 0015

[0071] Main instruments Dig Behv animal behavior analysis system, product of Shanghai Jiliang Software Technology Co., Ltd.; HIstolore BIOCVT type paraffin slicer, TP1020 type automatic dehydrator, HI1220 type roasting machine, HI1210 type spreading machine, EG1150H + C type tissue embedding machine, MSX11 type digital imaging system, all products of German Leica company. CX31 biological microscope, product of Olympus, Japan

[0072] Test methods Model establishment and grouping Ninety SPF-grade C57 mice with a body weight of 16.7 - 21.9 g were selected. Ten mice were randomly selected according to body weight as the normal control group. Except for the normal control group, the remaining mice were randomly subjected to various different stressors every day, including: restraint for 12 h, wet cage for 12 h, ice water swimming (4 °C) for 10 min, stroboscopic light for 12 h, 45-degree inclined cage for 12 h, noise for 30 min, circadian rhythm reversal, fasting for 12 h, water deprivation for 12 h, etc. The stimuli were randomly arranged every day, and the same stimulus could not appear continuously, so that the animals could not predict the given stimuli. There were 2 - 3 kinds of stimuli per day, and the modeling time lasted for 28 d. The schedule is shown in Table 5. After modeling, the mice were subjected to a sucrose preference test. According to the sucrose preference index, 70 mice were randomly divided into 7 groups, namely the model control group, the fluoxetine hydrochloride tablet group (10 mg / kg), the low, medium, and high-dose groups of Trillium tschonoskii Maxim. extract (25, 50, 100 mg / kg), the high-dose group of Trillium tschonoskii Maxim. water extract, and the high-dose group of Trillium tschonoskii Maxim. alcohol extract, with 10 mice in each group. The normal control group and the model control group were intragastrically administered with pure water, and the remaining groups of mice were intragastrically administered with the corresponding medicinal solutions, with the administration volume of 20 mL / kg. The medicine was administered once a day for 28 consecutive days. During the administration period, except for the normal control group, the remaining groups continued the modeling, and behavioral tests were conducted at the end of the administration. Chronic unpredictable stress (CUMS) mainly corresponds to major depressive disorder (MDD) clinically.

[0073]

[0074] 2 Detection indexes 2.1 Sucrose preference detection: The experiment was divided into an adaptation period, a training period, and a test period. During the adaptation period, the animals were given two bottles of 1% sucrose solution to adapt to sucrose drinking for 24 h. During the training period, the animals were given one bottle of 1% sucrose solution and one bottle of pure water for 24 h. During the test period, the animals were not fasted or water-deprived for 16 h before the test. Similarly, the animals were given one bottle of 1% sucrose solution and one bottle of pure water, and the test time was 24 h. To avoid the influence of position preference, the positions of the two bottles were exchanged during the test. After the test, the sucrose preference index was calculated. Sucrose preference index = sucrose consumption / (sucrose consumption + pure water consumption) × 100%.

[0075] 2.2 Open field test: When detecting, the mice were placed in the activity box. After adapting to the environment for 3 min, the Dig Behv animal behavior analysis system was used to record the spontaneous activity of the mice within 10 min.

[0076] 2.3 Forced swimming test: The JL Behv forced swimming video analysis system was used to detect the activity time of the mice within 5 min.

[0077] 2.4 Histopathological examination of hippocampus: After the behavioral tests, the hippocampal tissues of mice in each group were taken for HE staining for histopathological examination.

[0078] Experimental results 1 Effect of Trillium tschonoskii Maxim. extract 1 on the sucrose preference index of mice in the chronic unpredictable stress-induced depression model As shown in Table 5, compared with the normal control group, the sucrose preference index of mice in the model control group was significantly decreased ( P ≤0.01). Compared with the model control group, the sucrose preference indexes of mice in the low, medium, and high dose groups of Trillium tschonoskii Maxim. extract 1, the high dose of Trillium tschonoskii Maxim. alcohol extract, and the fluoxetine hydrochloride tablet group were significantly increased ( P ≤0.01 or P ≤0.05). Compared with the Trillium tschonoskii Maxim. water extract and high dose of alcohol extract groups, the sucrose preference indexes of mice in the low, medium, and high dose groups of Trillium tschonoskii Maxim. extract 1 and the fluoxetine hydrochloride tablet group were significantly increased ( P ≤0.05).

[0079]

[0080] Note: Compared with the normal control group, ++ P ≤0.01; compared with the model control group, ** P ≤0.01, compared with the Trillium tschonoskii Maxim. water extract and high dose of alcohol extract groups, #P≤0.05.

[0081] 2 Effect of Trillium tschonoskii Maxim. extract 1 on the open field activity of depressive model mice As shown in Table 6, compared with the normal control group, the activity time in the central area, the total activity time, and the distance traveled in the central area of mice in the model control group were significantly increased (P≤0.05 or P≤0.01), and the rest time was significantly decreased (P≤0.05). Compared with the model control group, the activity time in the central area of mice in the low, medium, and high dose groups of Trillium tschonoskii Maxim. extract 1 and the high dose of Trillium tschonoskii Maxim. alcohol extract group was significantly decreased ( P ≤0.01), and the rest time of mice in the medium and high dose groups of Trillium tschonoskii Maxim. extract 1 and the high dose of Trillium tschonoskii Maxim. alcohol extract group was significantly increased ( P ≤0.05). The total activity time of mice in the medium dose of Trillium tschonoskii Maxim. extract 1 and the high dose of Trillium tschonoskii Maxim. alcohol extract group was significantly decreased ( P ≤0.05). The activity time in the central area, the total activity time, and the distance traveled in the central area of mice in the fluoxetine hydrochloride tablet group were significantly decreased ( P ≤0.01), and the rest time was significantly increased ( P ≤0.01). Compared with the Trillium tschonoskii Maxim. water extract and high dose of alcohol extract groups, the activity time in the central area of mice in the medium dose group of Trillium tschonoskii Maxim. extract 1 was significantly decreased ( P ≤0.05), and the rest time was significantly increased ( P≤0.05). The distance of the middle area of the mice in the medium and high dose groups of Trillium tschonoskii Maxim. extract was significantly reduced ( P ≤0.05).

[0082]

[0083] Note: Compared with the normal control group, + P ≤0.05, ++ P ≤0.01; compared with the model control group, * P ≤0.05, ** P ≤0.01. Compared with the high dose groups of water extract and alcohol extract of Trillium tschonoskii Maxim., # P ≤0.05.

[0084] 3 Effect of Trillium tschonoskii Maxim. extract 1 on the immobility time of forced swimming in depressive mice As shown in Table 7, compared with the normal control group, the immobility time of forced swimming in the model control group of mice was significantly increased (P≤0.01). Compared with the model control group, the immobility time of forced swimming in the medium and high dose groups of Trillium tschonoskii Maxim. extract 1, the high dose group of alcohol extract of Trillium tschonoskii Maxim., and the fluoxetine hydrochloride tablet group of mice was significantly reduced ( P ≤0.05). Compared with the high dose groups of water extract and alcohol extract of Trillium tschonoskii Maxim., the immobility time of forced swimming in the medium and high dose groups of Trillium tschonoskii Maxim. extract 1 and the fluoxetine hydrochloride tablet group of mice was significantly reduced ( P ≤0.05).

[0085]

[0086] Note: Compared with the normal control group, ++ P ≤0.01; compared with the model control group, * P ≤0.05. Compared with the high dose groups of water extract and alcohol extract of Trillium tschonoskii Maxim., # P ≤0.05.

[0087] 4 Effect of Trillium tschonoskii Maxim. extract 1 on the histopathology of the hippocampus in mice The results of the effect of Trillium tschonoskii Maxim. extract 1 on the histopathology of the hippocampus in mice are as Figure 3As shown in the figure: In the figure, A is the normal control group; B is the model control group; C is the fluoxetine hydrochloride group; D is the low-dose group of Trillium tschonoskii Maxim. extract 1; E is the medium-dose group of Trillium tschonoskii Maxim. extract 1; F is the high-dose group of Trillium tschonoskii Maxim. extract 1; G is the high-dose group of Trillium tschonoskii Maxim. water extract; H is the high-dose group of Trillium tschonoskii Maxim. alcohol extract. It can be seen from the figure that in the hippocampal tissue of mice in the normal control group, the morphology of neurons, glial cells, and hippocampal pyramidal cells were normal; in the hippocampal tissue of mice in the model control group, some pyramidal cells were arranged sparsely and disorderly, some pyramidal cells were necrotic, and there was interstitial edema. In the hippocampal tissue of mice in the low-dose group of Trillium tschonoskii Maxim. extract 1, some pyramidal cells were necrotic and there was interstitial edema; in the medium- and high-dose groups of mice, some hippocampal pyramidal cells were arranged closely; there was a small amount of interstitial edema. In the hippocampal tissue of mice in the high-dose group of Trillium tschonoskii Maxim. water extract, some pyramidal cells were arranged sparsely and disorderly, some pyramidal cells were necrotic, and there was interstitial edema. In the hippocampal tissue of mice in the high-dose group of Trillium tschonoskii Maxim. alcohol extract, some pyramidal cells were arranged sparsely and disorderly, and there was interstitial edema. In the hippocampal tissue of mice in the fluoxetine hydrochloride group, the hippocampal pyramidal cells were arranged closely, and there was a small amount of interstitial edema.

[0088] Summary of the experiment The results showed that the low, medium, and high doses of Trillium tschonoskii Maxim. extract 1 could significantly increase the sucrose preference index of mice and significantly reduce the activity time of mice in the central area of the open field. The medium and high doses could significantly reduce the immobility time of mice in the forced swimming test. The medium dose could significantly increase the rest time of mice, significantly reduce the activity time, and could significantly improve the pathological changes of the hippocampal tissue of mice. Compared with the high-dose groups of Trillium tschonoskii Maxim. water extract and alcohol extract, the low, medium, and high doses of Trillium tschonoskii Maxim. extract 1 could significantly increase the sucrose preference index of mice. The medium dose of Trillium tschonoskii Maxim. extract 1 could significantly reduce the central activity time of mice, and the rest time was significantly increased. The medium and high doses of Trillium tschonoskii Maxim. extract 1 could significantly reduce the distance of mice in the central area. The medium and high doses of Trillium tschonoskii Maxim. extract 1 could significantly reduce the immobility time of mice in the forced swimming test. The results suggested that Trillium tschonoskii Maxim. extract 1 could significantly improve the depression-like behavior of mice induced by chronic unpredictable stress, had a significant antidepressant effect, and the antidepressant effect was better than that of Trillium tschonoskii Maxim. water extract and alcohol extract.

[0089] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. The use of Trillium extract in the preparation of a drug for treating depression, characterized in that: The extraction method of the Trillium extract comprises the following steps: S1. Add ethanol solution to Trillium in a ratio of 1:10-12, and reflux extraction at 80-90° C. to obtain an extract; S2. Concentrate the extract once, then add a macroporous resin for elution to obtain an eluate; during the elution process, first use 5-10BV of an ethanol aqueous solution with a mass concentration of 10-20% for elution, and the elution rate is 2-3BV / h; then use 5-10BV of an ethanol aqueous solution with a mass concentration of 70-80% for elution, and the elution rate is 2-3BV / h, and the eluate of 70-80% ethanol aqueous solution is collected for subsequent centrifugation; concentrate twice and freeze-dry to obtain the Trillium extract.

2. The use according to claim 1, characterized in that: The ethanol solution is an ethanol aqueous solution with a mass concentration of 40%-60%; the number of reflux extractions is 2-3 times.

3. The use according to claim 1, characterized in that: The primary concentration is to concentrate the extract to a concentration 3-5 times of the original concentration.

4. The use according to claim 1, characterized in that: The macroporous resin is a styrene-type non-polar copolymer macroporous resin.

5. The use according to claim 4, characterized in that: The macroporous resin is HPD-100 macroporous resin, and the added amount is 100-300g.

6. The use according to claim 1, characterized in that: During the elution process, the height ratio of the sample layer to the resin layer is 1-2:1-2.

7. The use according to any one of claims 1 to 6, characterized in that: The eluate obtained by eluting with 10-20% ethanol aqueous solution is impurities.

8. The use according to claim 1, characterized in that: The freeze-drying process is: the cold trap temperature is -47 to -48 degrees and the vacuum degree is 13 to 15 Pa.

9. The use according to claim 1, characterized in that: The medicine also includes other active ingredients for treating depression or other pharmaceutically acceptable excipients.

10. The use according to claim 9, characterized in that: The dosage form of the medicine is decoction, pill, capsule, tablet, powder or granule.

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