Application of Trillium Extract in the Preparation of Drugs for Treating Depression

Abstract

This invention relates to the field of depression treatment technology, and in particular to the application of Trillium truncatum extract in the preparation of drugs for treating depression. The extraction method of the Trillium truncatum extract includes the following steps: S1, adding ethanol solution to Trillium truncatum at a material-to-liquid ratio of 1:10-12, and refluxing at 80-90℃ to obtain an extract; S2, concentrating the extract once, then adding macroporous resin for elution to obtain an eluent; centrifuging, concentrating a second time, and freeze-drying the eluent to obtain the Trillium truncatum extract. In this invention, the use of Trillium truncatum extract as a drug for treating depression can significantly alleviate symptoms of depression and psychogenic depression in mice. Furthermore, since Trillium truncatum extract is a natural substance, it has no toxic side effects, and its price is far lower than existing Western medicines for treating depression, making it convenient for widespread application in the treatment of depression.

Description

Technical Field

[0001] This invention relates to the field of depression treatment technology, and in particular to the application of Trillium extract in the preparation of drugs for treating depression. Background Technology

[0002] Depression, also known as depressive disorder, is a mental disorder with a high incidence, high clinical cure rate but low treatment acceptance rate and high relapse rate. Clinically, depressive disorders can be classified into mild, moderate, and severe based on 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:

[0003] A. Five or more of the following symptoms appear within the same two-week period, showing a change in function compared to previous function (at least one of which is (1) depressed mood or (2) loss of interest or pleasure):

[0004] 1. Depressed mood almost every day and for most of the day, which can be reported subjectively (e.g., feeling sad, empty, hopeless) or observed by others (e.g., manifested as crying).

[0005] 2. Almost every day and for most of the day, there is a significant decrease in interest or pleasure in all or almost all activities (this can be either a subjective statement or an observation).

[0006] 3. Significant weight loss or gain without dieting (e.g., weight change exceeding 5% of original weight within a month), or decreased or increased appetite almost daily (Note: Children may exhibit failure to reach the standard weight).

[0007] 4. I either suffer from insomnia or sleep excessively almost every day.

[0008] 5. Almost daily psychomotor agitation or sluggishness (visible to others, not just subjectively experienced as restlessness or lethargy).

[0009] 6. Feeling tired or lacking energy almost every day.

[0010] 7. Feeling worthless almost every day, or feeling excessive and inappropriate guilt (to the point of delusions, and not just feeling guilty or remorseful because of the illness).

[0011] 8. Almost every day there is a decline in thinking ability or inability to concentrate, or indecisiveness (this can be a subjective statement or an observation by others).

[0012] 9. Recurring thoughts of wanting to die (not just fear of death), recurring suicidal ideation without a specific plan, or some kind of suicide attempt, or some specific plan to carry out suicide.

[0013] B. These symptoms cause clinically significant distress or lead to impairment in social, occupational, or other important functions.

[0014] C. These symptoms cannot be attributed to the physiological effects of a substance or other physical illnesses.

[0015] Depression is defined as meeting 5 or more of the diagnostic criteria A.

[0016] Meeting all of the diagnostic criteria A, B, and C constitutes a major depressive episode.

[0017] Antidepressants can be classified into three categories based on their mechanism of action and time of introduction: First-generation antidepressants, mainly non-selective monoamine oxidase inhibitors (MAOIs) and tricyclic antidepressants, such as phenelzine; second-generation antidepressants, mainly reversible and selective MAOIs and tetracyclic antidepressants, such as moclobemide; and third-generation antidepressants, mainly selective serotonin reuptake inhibitors (SSRIs), selective norepinephrine reuptake inhibitors (NRRIs), and inhibitors of both neurotransmitters, such as fluoxetine and mirtazapine. Drugs with these mechanisms are currently more widely used clinically. In addition to drug treatment, physical therapy is also a common method for treating depression, such as electroconvulsive therapy (ECT), sleep deprivation, and psychotherapy.

[0018] First-generation antidepressants have numerous adverse reactions and have been withdrawn from first-line clinical use. Currently, second- and third-generation antidepressants are more commonly used clinically. However, current drug-based treatments suffer from poor patient adherence due to adverse reactions. For moderate to severe depression, drug therapy is not highly effective, and relapse is common. Some patients even experience significant central nervous system side effects during drug treatment. Physical therapy also has drawbacks; for example, electroconvulsive therapy (ECT) is primarily used for patients with poor medication adherence. It requires pre-anesthesia before electrical stimulation, carrying a certain risk of death during anesthesia. Furthermore, ECT can severely impair the cognitive function of depressed patients. Summary of the Invention

[0019] The purpose of this invention is to provide the application of Trillium extract in the preparation of drugs for treating depression.

[0020] To achieve the above objectives, the present invention adopts the following technical solution:

[0021] The application of Trillium truncata extract in the preparation of drugs for treating depression, wherein the extraction method of Trillium truncata extract includes the following steps:

[0022] S1. Add ethanol solution to Trillium truncatum at a material-to-liquid ratio of 1:10-12, and reflux extract at 80-90℃ to obtain the extract.

[0023] S2. The extract was concentrated once, and then eluted with macroporous resin to obtain the eluent. During the elution process, 5-10 BV of 10-20% ethanol aqueous solution was used for elution at a rate of 2-3 BV / h. Then, 5-10 BV of 70-80% ethanol aqueous solution was used for elution at a rate of 2-3 BV / h. The eluent of 70-80% ethanol aqueous solution was collected for subsequent centrifugation. The extract was concentrated a second time and freeze-dried to obtain the Trillium truncatum extract.

[0024] In one preferred embodiment, the feed-to-liquid ratio is expressed as raw material mass: solvent volume. For example, a feed-to-liquid ratio of 1:10 g / mL means that 1 part by mass of raw material corresponds to 10 parts by volume of solvent.

[0025] In one preferred embodiment, the ethanol solution is an aqueous ethanol solution with a mass concentration of 40%-60%.

[0026] In one preferred embodiment, the reflux extraction is performed 2-3 times.

[0027] In one preferred embodiment, the concentration is performed by concentrating the extract to a concentration 3-5 times that of the original.

[0028] In one preferred embodiment, the macroporous resin is a styrene-type nonpolar copolymer macroporous resin.

[0029] In one preferred embodiment, the macroporous resin is HPD-100 macroporous resin, and the amount added is 100-300g.

[0030] In one preferred embodiment, during elution, the height ratio of the sample layer to the resin layer is 1-2:1-2.

[0031] In one preferred embodiment, during the elution process, elution is first performed using 5-10 BV of an ethanol aqueous solution with a mass concentration of 10-20% at a elution rate of 2-3 BV / h; then elution is performed using 5-10 BV of an ethanol aqueous solution with a mass concentration of 70-80% at a elution rate of 2-3 BV / h, and this eluent is collected for subsequent centrifugation.

[0032] The eluent obtained by elution with an ethanol aqueous solution of 10-20% by mass concentration is an impurity and should be discarded.

[0033] In one preferred embodiment, the centrifugation speed is 3000-4000 r / min and the time is 10-15 min.

[0034] In one preferred embodiment, the secondary concentration involves concentrating the extract to a concentration 3-5 times the original concentration.

[0035] In one preferred embodiment, the freeze-drying process is as follows: the cold trap temperature is -47 to -48 degrees Celsius, and the vacuum degree is 13 to 15 Pa.

[0036] In one preferred embodiment, the drug also includes other active ingredients for treating depression.

[0037] In one preferred embodiment, the drug further includes other pharmaceutically acceptable excipients.

[0038] In one preferred embodiment, the dosage form of the drug is a decoction, pill, capsule, tablet, powder, or granule.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] In this invention, Trillium extract is used as a drug for treating depression. It can significantly alleviate depression and psychogenic depressive symptoms in mice. At the same time, since Trillium extract is a natural substance, it has no toxic side effects and its price is much lower than that of existing Western medicines for treating depression, making it easy to be widely used in the treatment of depression. Attached Figure Description

[0041] Figure 1 The appearance characteristics of Trillium;

[0042] Figure 2 A flowchart illustrating the process for extracting Trillium oxypetalum extract;

[0043] Figure 3 HE staining image of the effect of Trillium extract 1 on the pathological characteristics of mouse hippocampus tissue in Example 1;

[0044] 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 extract 1; E is the medium-dose group of Trillium extract 1; F is the high-dose group of Trillium extract 1; G is the high-dose group of Trillium water; H is the high-dose group of Trillium alcohol. Detailed Implementation

[0045] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0046] Example 1

[0047] Method 1: Extraction, enrichment and purification of Trillium extract 1

[0048] Experimental materials:

[0049] Trillium tschonoskii Maxim. is a perennial herb belonging to the genus Trillium in the family Veratraceae. It has a sweet taste, neutral properties, and is slightly toxic. It has been included in the "Standards for Chinese Medicinal Materials of Hubei Province".

[0050] Trillium, such as Figure 1 Place of origin: Northeast China; Product batch number: 241101; Production date: November 20, 2024.

[0051] Experimental procedure:

[0052] according to Figure 2 The process flow diagram shown is extracted as follows:

[0053] 1. Extraction

[0054] Take 500g of Trillium and add 50% ethanol aqueous solution at a material-to-liquid ratio of 1:12g / mL. Reflux and extract twice at 80-90℃, 2 hours each time.

[0055] 2. Enrichment of HPD-100 (styrene-type nonpolar copolymer) macroporous resin

[0056] After concentrating to 1 / 3 of its original volume, 200g of HPD-100 macroporous resin was added, and the mixture was evaporated on a rotary evaporator until it did not stick to the walls and the particles were clearly separated. Then, the sample layer and the blank resin layer (filler layer) were packed into a column with a 1:1 thickness ratio. Elution was performed with 5 BV (1 BV = 2.1 L) of a 20% ethanol aqueous solution at a flow rate of 3 BV / h; this eluent was discarded as impurities. Next, elution was performed with 5 BV of a 70% ethanol aqueous solution at a flow rate of 3 BV / h, and this eluent was collected.

[0057] 3. Concentration process

[0058] The 70% ethanol fraction was concentrated using a rotary evaporator, followed by centrifugation at 3500 r / min for 10 min to obtain insoluble matter and a centrifuged liquid. The centrifuged liquid was further concentrated using a rotary evaporator until it reached a three-fold concentration, with a volume of approximately 200 mL. The insoluble matter and concentrate were then frozen at -80°C and subsequently freeze-dried in a freeze dryer at a cold trap temperature of -47 to -48°C and a vacuum degree of 13 to 15 Pa. The final product was approximately 39.67 g of powdered Trillium truncatum extract 1.

[0059] Method 2: Preparation of Trillium water extract

[0060] Preparation method of Trillium aqueous extract: 500g of Trillium was decocted twice with water at a material-to-liquid ratio of 1:12g / mL. The filtrates were combined and concentrated. After drying in an oven, 92.85g of Trillium aqueous extract was obtained.

[0061] Method 3: Preparation of Trillium truncatum alcohol extract

[0062] Preparation method of Trillium truncatum ethanol extract: Weigh 500g of Trillium truncatum and add it to a round-bottom flask at a solid-liquid ratio of 1:12g / mL. Add 75% ethanol as the extraction solvent. Fix the round-bottom flask on a reflux evaporator and reflux at 80℃ for 3 hours. After extraction, filter the extract and transfer it to a rotary evaporator. Concentrate the extract under reduced pressure, generally at a temperature of 40-60℃, until it is reduced to 1 / 3 of its original volume, to obtain a concentrated Trillium truncatum ethanol extract. Transfer the concentrated extract to a petri dish and dry it in a low-temperature drying oven to obtain 31.28g of Trillium truncatum ethanol extract solid powder.

[0063] 1. Effects on reserpine-induced mouse depression model

[0064] Test sample

[0065] Trillium, batch number: 20240109, expiry date: September 2022, provided by Hunan Prima Pharmaceutical Research Center Co., Ltd.

[0066] Reference substances

[0067] Clomipramine hydrochloride tablets, batch number: LM202203, product of Jiangsu Enhua Pharmaceutical Co., Ltd.

[0068] experimental animals

[0069] Ninety SPF-grade ICR mice, weighing 15.2–28.8 g, half male and half female, were purchased from Changsha Tianqin Biotechnology Co., Ltd., with laboratory animal production license number: SCXK (Xiang) 2019-0013 and laboratory animal quality certificate number: 430726221100110776. They were housed in Area D of the Barrier Environment Laboratory of Hunan Prima Pharmaceutical Research Center Co., Ltd., with laboratory animal use license number: SYXK (Xiang) 2020-0015.

[0070] Main reagents

[0071] Reserpine Injection, specification: 1mL:1mg, batch number: 2006041, Tianjin Jinyao Pharmaceutical Co., Ltd.

[0072] Main instruments

[0073] AUY220 analytical balance, AUW-220D analytical balance, ME2002E / 02 electronic balance, Mettler Company products.

[0074] Test methods

[0075] Grouping and Dosing

[0076] Ninety qualified ICR mice, weighing 18.2–21.4 g, with half males and half females, were randomly divided into four groups according to their weight: a normal control group, a model control group, a clomipramine hydrochloride tablet group (9.75 mg / kg), low- and high-dose Trillium truncatum water extract groups (70 and 280 mg / kg), low- and high-dose Trillium truncatum alcohol extract groups (70 and 280 mg / kg), and low- and high-dose Trillium truncatum extract groups (70 and 280 mg / kg), with 10 mice in each group, half males and half females. The normal control group and the model control group were given pure water by gavage at a volume of 20 mL / kg. The other groups of mice were given the corresponding drug solution by gavage. The drugs were administered once a day for 7 consecutive days. One hour after the last administration, except for the normal control group mice, which were injected intraperitoneally with 0.9% sodium chloride injection, the other groups of mice were injected intraperitoneally with reserpine 2 mg / kg at a volume of 20 mL / kg. Eyelid closure and anal temperature were measured after the injection.

[0077] Experimental results

[0078] 1. Effects of different extraction methods of Trillium truncata on eyelid closure and rectal temperature in reserpine-induced depression model mice

[0079] Compared with the model control group, mice in the low-dose and high-dose groups of Trillium truncatum aqueous extract, the low-dose and high-dose groups of Trillium truncatum ethanol extract, and the low-dose and high-dose groups of Trillium truncatum extract 1 showed a significant decrease in eyelid closure. P ≤0.01), rectal temperature significantly increased ( P≤0.01). Compared with the low-dose group of Trillium extract 1, the eyelid closure of mice in the low-dose and high-dose groups of water extract and the low-dose and high-dose groups of alcohol extract was significantly increased ( P ≤0.05 or P ≤0.01), the anal temperature of mice in the high-dose water extract group, the low-dose alcohol extract group, and the high-dose alcohol extract group was significantly reduced ( P ≤0.05%. Compared with the high-dose group of Trillium extract 1, the eyelid closure of mice in the low-dose and high-dose groups of water extract and the low-dose and high-dose groups of alcohol extract were significantly increased ( P ≤0.05 or P ≤0.01), rectal temperature was significantly reduced. Ptosis was significantly reduced in mice in the clomipramine hydrochloride tablet group ( P ≤0.01), rectal temperature significantly increased ( P ≤0.01).

[0080]

[0081] 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 extract 1 # P ≤0.05, ## P ≤0.01; compared with the high-dose group of Trillium extract 1 ▲ P ≤0.05, ▲▲ P ≤0.01.

[0082] Experiment Summary

[0083] Trillium extract 1, at both low and high doses, significantly reduced eyelid closure and significantly increased anal temperature in mice. The results showed that Trillium extract 1 had a significant antidepressant effect, which was significantly higher than that of the aqueous and alcoholic extracts of Trillium.

[0084] Example 2

[0085] Effects of Trillium Extract 1 on an Olfactory Bulb Removal-Induced Rat Depression Model

[0086] Test materials

[0087] Test sample

[0088] Trillium extract 1, batch number: 240201, expiry date: 2026.02.15, extracted from Example 1.

[0089] Reference substances

[0090] Fluoxetine Hydrochloride, batch number: 202410311, specification: 10 mg / tablet, expiration date: 2024-10-30, product of Changzhou Siyao Pharmaceutical Co., Ltd.

[0091] Experimental animals

[0092] 70 SPF-grade male WKY rats, weighing 151.8 - 218.6 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The production license number of experimental animals is SCXK (Beijing) 2021-0006, and the quality certificate number of experimental animals is 110011231107853326. They were housed in Area D of the barrier environment laboratory of Hunan Prima Pharmaceutical Research Center. The license number for the use of experimental animals is SYXK (Hunan) 2020-0015.

[0093] Main instruments

[0094] JLBehv Animal Behavior Video Analysis System, product of Shanghai Jiliang Software Technology Co., Ltd.; ALC-H Electric Digital Animal Brain Stereotaxic Instrument, product of Shanghai Aolte Biotechnology Co., Ltd.; ALC-CED Type Animal Skull Drill: product of Shanghai Aolte Biotechnology Co., Ltd.

[0095] Test methods

[0096] Animal modeling

[0097] 90 SPF-grade male WKY rats with qualified quarantine, weighing 151.8 - 218.6 g, were 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 the brain stereotaxic instrument. The skin of the rats' heads was disinfected with iodophor and cut open to expose 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 bone drill, and the olfactory bulbs were destroyed with a special round blunt needle and then aspirated. After the rats' wounds were hemostatic packed, sealed with bone wax, and anti-infection treatment was carried out, the skin was sutured. Olfactory bulbectomy-induced depression mainly corresponds to major depressive disorder (MDD) clinically.

[0098] Grouping and administration

[0099] After 14 days of single-cage feeding following surgery, surviving rats were randomly divided into 7 groups based on their sucrose preference index: a model control group, a fluoxetine hydrochloride tablet group, low, medium, and high dose groups of Trillium truncatum extract, a high dose group of Trillium truncatum water, and a high dose group of Trillium truncatum alcohol, with 10 rats in each group. Rats in the normal control and model control groups were administered pure water by gavage, while rats in the other groups were administered the corresponding drug solution by gavage. The drug volume was 10 mL / kg, once daily, for 45 consecutive days. Relevant behavioral tests were performed after the last administration.

[0100] detection indicators

[0101] 1. Sugar Water Preference Test: The experiment was divided into an adaptation period, a training period, and a testing period. During the adaptation period, animals were given two bottles of 1% sucrose solution to allow them to adapt to drinking sucrose for 24 hours. During the training period, animals were given one bottle of 1% sucrose solution and one bottle of pure water for 24 hours. During the testing period, animals were deprived of food and water for 16 hours before the test. Again, animals were given one bottle of 1% sucrose solution and one bottle of pure water for 24 hours. To avoid the influence of positional preference, the positions of the two bottles were swapped during the test. At the end of the test, the sugar water preference index was calculated as: Sugar water preference index = (Sugar water consumption / (Sugar water consumption + Pure water consumption)) × 100%.

[0102] 2. Forced swimming test: The JL Behv forced swimming video analysis system was used to detect the resting time and activity time of rats within 5 minutes.

[0103] Experimental results

[0104] 1. Effect of Trillium Extract 1 on Saccharide Preference Index in a Rats with Olfactory Bulb Removal and Depression

[0105] As shown in Table 2, compared with the normal control group, the sucrose preference index of rats in the model control group was significantly decreased (P≤0.01). Compared with the model control group, the sucrose preference index of rats in the low, medium, and high dose groups of Trillium truncatum extract 1 was significantly increased (P≤0.01). P ≤0.05 or P ≤0.01). Compared with the high-dose groups of Trillium truncatum water extract and alcohol, the saccharide preference index of rats in the medium and high dose groups of Trillium truncatum extract 1 was significantly increased ( P ≤0.05).

[0106]

[0107] 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 Trillium truncatum water extract and alcohol, # P ≤0.05.

[0108] 2. Effect of Trillium Extract 1 on Immobility Time in Rats During Forced Swimming

[0109] As shown in Table 3, compared with the normal control group, the forced swimming immobility time of rats in the model control group was significantly increased (P≤0.01). Compared with the model control group, the forced swimming immobility time of rats in the medium and high dose groups of Trillium extract 1 was significantly decreased (P≤0.05 or P≤0.01). The forced swimming immobility time of rats in the fluoxetine hydrochloride tablet group was significantly decreased ( P ≤0.01). Compared with the high-dose groups of Trillium truncatum water extract and alcohol, the forced swimming immobility time of rats in the high-dose group of Trillium truncatum extract 1 was significantly reduced ( P ≤0.01). Compared with the high-dose trillium ol group, the forced swimming immobility time of rats treated with high-dose trillium extract 1 was significantly reduced ( P ≤0.01).

[0110]

[0111] 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 Trillium truncatum water extract and alcohol, ## P ≤0.01.

[0112] Experiment Summary

[0113] Trillium extract 1 was tested at low, medium, and high doses. Medium and high doses of Trillium extract 1 significantly reduced the immobility time during forced swimming. Compared with the increased dose group of trillium alcohol, medium and high doses of Trillium extract 1 significantly increased the sucrose preference index in rats. High doses of Trillium extract 1 significantly reduced the immobility time during forced swimming in rats. The results showed that Trillium extract 1 significantly improved depressive behavior in rats, and its antidepressant effect was superior to that of Trillium aqueous extract and Trillium alcohol extract.

[0114] Example 3

[0115] Effects of Trillium Extract 1 on a Mouse Model of Depression Under Chronic Unpredictable Stress

[0116] Test materials

[0117] test substance

[0118] Trillium extract 1, batch number: 240201, expiry date: 2026.02.15, prepared according to the example.

[0119] Reference substances

[0120] Fluoxetine hydrochloride tablets, batch number: 202410311, specification: 10mg / tablet, expiry date: 2024.10.30, product of Changzhou No.4 Pharmaceutical Co., Ltd.

[0121] experimental animals

[0122] Eighty SPF-grade male C57 mice, weighing 16.7-21.9g, were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., with Experimental Animal Production License No.: SCXK (Xiang) 2019-0004 and Experimental Animal Quality Certificate No.: 430727231101501437. They were housed in Area D of the Barrier Environment Laboratory of Hunan Prima Pharmaceutical Research Center Co., Ltd., with Experimental Animal Use License No.: SYXK (Xiang) 2020-0015.

[0123] Main instruments

[0124] The Dig Behv animal behavior analysis system is a product of Shanghai Jiliang Software Technology Co., Ltd.; the HIstolore BIOCVT paraffin microtome, TP1020 fully automatic dehydrator, HI1220 slide roaster, HI1210 slide spreader, EG1150H+C tissue embedding machine, and MSX11 digital imaging system are all products of Leica GmbH, Germany. The CX31 biological microscope is a product of Olympus, Japan.

[0125] Test methods

[0126] Modeling and Grouping

[0127] Ninety qualified SPF-grade C57 mice, weighing 16.7-21.9g, were selected. Ten mice were randomly selected as the normal control group based on their weight. Except for the normal control group, the remaining mice were randomly subjected to various stressors every day, including: restraint for 12 hours, wet cage for 12 hours, swimming in ice water (4℃) for 10 minutes, strobe light for 12 hours, 45-degree inclined cage for 12 hours, noise for 30 minutes, day-night reversal, fasting for 12 hours, and water deprivation. The stressors were randomly arranged every day, and the same stressor could not be given consecutively so that the animals could not predict the stressors given. Two to three types of stressors were given every day, and the modeling period lasted for 28 days. The timetable is shown in Table 5. After modeling, mice underwent a saccharide preference test. Based on the saccharide preference index, 70 mice were randomly selected and divided into 7 groups: a model control group, a fluoxetine hydrochloride tablet group (10 mg / kg), low, medium, and high dose groups of Trillium extract (25, 50, and 100 mg / kg), a high-dose Trillium water group, and a high-dose Trillium alcohol group (n=10 per group). The normal control and model control groups were administered pure water by gavage, while the other groups were administered the corresponding drug solution by gavage at a volume of 20 mL / kg, once daily for 28 consecutive days. During the administration period, except for the normal control group, the modeling process continued in the other groups. Behavioral tests were performed at the end of the administration period. Chronic unpredictable stress (CUMS) clinically mainly corresponds to major depressive disorder (MDD).

[0128]

[0129] 2 detection indicators

[0130] 2.1 Sugar Water Preference Test: The experiment was divided into an adaptation period, a training period, and a testing period. During the adaptation period, animals were given two bottles of 1% sucrose solution to allow them to adapt to sucrose drinking water for 24 hours. During the training period, animals were given one bottle of 1% sucrose solution and one bottle of pure water for 24 hours. During the testing period, animals were deprived of food and water for 16 hours before the test. Again, animals were given one bottle of 1% sucrose solution and one bottle of pure water for 24 hours. To avoid the influence of positional preference, the positions of the two bottles were interchanged during the test. At the end of the test, the sugar water preference index was calculated as: Sugar water preference index = (Sugar water consumption / (Sugar water consumption + Pure water consumption)) × 100%.

[0131] 2.2 Open field test: During the test, the mice were placed in an activity box and allowed to adapt to the environment for 3 minutes. Then, the Dig Behv animal behavior analysis system was used to record the mice's spontaneous activities within 10 minutes.

[0132] 2.3 Forced swimming test: The activity time of mice within 5 minutes was detected using the JL Behv forced swimming video analysis system.

[0133] 2.4 Hippocampal histopathological examination: After the behavioral test, the hippocampal tissue of each group was taken for HE staining and histopathological examination.

[0134] Experimental results

[0135] 1. Effect of Trillium Extract 1 on Saccharide Preference Index in Mice with Chronic Unpredictable Stress Depression Model

[0136] As shown in Table 5, compared with the normal control group, the sucrose preference index of mice in the model control group was significantly reduced ( P ≤0.01). Compared with the model control group, mice in the low, medium, and high doses of Trillium extract 1, the increased dose of Trillium alcohol, and the fluoxetine hydrochloride tablet group showed significantly increased sucrose preference index ( ). P ≤0.01 or P ≤0.05). Compared with the high-dose groups of Trillium truncatum water extract and alcohol, the sucrose preference index of mice in the low, medium, and high dose groups of Trillium truncatum extract 1 and the fluoxetine hydrochloride tablet group was significantly increased ( P ≤0.05).

[0137]

[0138] Note: Compared with the normal control group, ++ P ≤0.01; compared with the model control group, ** P ≤0.01, compared with the high-dose groups of Trillium truncatum water extract and alcohol, #P≤0.05.

[0139] 2. Effects of Trillium Extract 1 on Open Field Activity in Depressed Mice

[0140] As shown in Table 6, compared with the normal control group, the central activity time, activity duration, and central distance traveled by mice in the model control group were significantly increased (P≤0.05 or P≤0.01), while the rest time was significantly decreased (P≤0.05). Compared with the model control group, the central activity time of mice in the low, medium, and high dose groups of Trillium extract 1 and the high dose group of Trillium alcohol were significantly reduced (P≤0.05). P ≤0.01), the resting time of mice in the medium and high dose groups of Trillium extract 1 and the increased dose group of Trillium alcohol was significantly increased ( P ≤0.05). Medium doses of Trillium extract and increased doses of Trillium alcohol significantly reduced activity time in mice. P ≤0.05). The central region activity time, activity duration, and central region distance were significantly reduced in mice in the fluoxetine hydrochloride tablet group. P ≤0.01), rest time increased significantly ( P ≤0.01). Compared with the high-dose groups of Trillium water extract and alcohol, the central activity time of mice in the medium-dose group of Trillium extract 1 was significantly reduced ( P≤0.05), rest time increased significantly ( P ≤0.05). The central region distance in mice was significantly reduced in the medium and high dose groups of Trillium extract 1 (≤0.05). P ≤0.05).

[0141]

[0142] 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 Trillium truncatum water extract and alcohol, # P ≤0.05.

[0143] 3. Effect of Trillium Extract 1 on the Immobility Time During Forced Swimming in Depressed Mice

[0144] As shown in Table 7, compared with the normal control group, the forced swimming immobility time of mice in the model control group was significantly increased (P≤0.01). Compared with the model control group, the forced swimming immobility time of mice in the medium and high doses of Trillium extract 1, the increased dose group of Trillium alcohol, and the fluoxetine hydrochloride tablet group was significantly reduced (P≤0.01). P ≤0.05%. Compared with the high-dose groups of Trillium truncatum water extract and alcohol, the forced swimming immobility time of mice in the medium and high dose groups of Trillium truncatum extract 1 and the fluoxetine hydrochloride tablet group was significantly reduced. P ≤0.05).

[0145]

[0146] 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 Trillium truncatum water extract and alcohol, # P ≤0.05.

[0147] 4. Effects of Trillium Extract 1 on Hippocampal Histopathology in Mice

[0148] The effects of Trillium extract 1 on mouse hippocampal histopathology are as follows: Figure 3As shown 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 truncatum extract 1; E is the medium-dose group of Trillium truncatum extract 1; F is the high-dose group of Trillium truncatum extract 1; G is the high-dose group of Trillium truncatum water; H is the high-dose group of Trillium truncatum alcohol. From the figure, it can be seen that in the normal control group, no abnormalities were observed in the morphology of neurons, glial cells, and hippocampal pyramidal cells in the hippocampus tissue of mice. In the model control group, some pyramidal cells were sparsely and disordered, some pyramidal cells were necrotic, and interstitial edema was observed in the hippocampus tissue of mice. In the low-dose group of Trillium truncatum extract 1, some pyramidal cells were necrotic and interstitial edema were observed in the hippocampus tissue of mice. In the medium and high-dose groups, some pyramidal cells were tightly arranged, and a small amount of interstitial edema was observed in the hippocampus tissue of mice. In the high-dose group of Trillium truncatum water, some pyramidal cells were sparsely and disordered, some pyramidal cells were necrotic, and interstitial edema was observed in the hippocampus tissue of mice. In the high-dose group of Trillium truncatum alcohol, some pyramidal cells were sparsely and disordered, and interstitial edema was observed in the hippocampus tissue of mice. In mice in the fluoxetine hydrochloride group, hippocampal pyramidal cells were found to be tightly packed with a small amount of intercellular edema.

[0149] Experiment Summary

[0150] The results showed that low, medium, and high doses of Trillium truncata extract 1 significantly increased the saccharide preference index in mice and significantly reduced the activity time in the central region of mice in open field. Medium and high doses significantly reduced the immobility time during forced swimming in mice. The medium dose significantly increased rest time, significantly reduced activity time, and significantly improved the pathological changes in the hippocampus of mice. Compared with the high-dose groups of Trillium truncata water extract and alcohol extract, low, medium, and high doses of Trillium truncata extract 1 significantly increased the saccharide preference index in mice. The medium dose of Trillium truncata extract 1 significantly reduced the central activity time and significantly increased the rest time in mice. Medium and high doses of Trillium truncata extract 1 significantly reduced the central region travel distance in mice. Medium and high doses of Trillium truncata extract 1 significantly reduced the immobility time during forced swimming in mice. These results suggest that Trillium truncata extract 1 can significantly improve depressive-like behavior induced by chronic unpredictable stress in mice, exhibiting a significant antidepressant effect, and its antidepressant effect is superior to that of Trillium truncata water extract and alcohol extract.

[0151] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. The application of Trillium extract in the preparation of drugs for treating depression, characterized in that, The extraction method of the Trillium extract includes the following steps: S1. Add ethanol solution to Trillium tsao-ko at a material-to-liquid ratio of 1:10-12, and reflux extract at 80-90℃ to obtain extract; the ethanol solution is an aqueous ethanol solution with a mass concentration of 40%-60%; the reflux extraction is performed 2-3 times. S2. The extract is concentrated once, and then eluted with macroporous resin, which is a styrene-type nonpolar copolymer macroporous resin, specifically HPD-100 macroporous resin, at a concentration of 100-300g, to obtain the eluent. During elution, elution is first performed with 5-10 BV of 10-20% ethanol aqueous solution at a rate of 2-3 BV / h; then elution is performed with 5-10 BV of 70-80% ethanol aqueous solution at a rate of 2-3 BV / h. The eluent of the 70-80% ethanol aqueous solution is collected for subsequent centrifugation; the extract is then concentrated a second time and freeze-dried to obtain the Trillium truncata extract.

2. The application according to claim 1, characterized in that, One-time concentration means concentrating the extract to a concentration 3-5 times the original concentration.

3. The application 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.

4. The application according to any one of claims 1-3, characterized in that, The eluent obtained by elution with an ethanol aqueous solution of 10-20% by mass concentration is an impurity.

5. The application according to claim 1, characterized in that, The freeze-drying process involves a cold trap temperature of -47 to -48 degrees Celsius and a vacuum level of 13 to 15 Pa.

6. The application according to claim 1, characterized in that, The drug also includes other pharmaceutically acceptable excipients.

7. The application according to claim 6, characterized in that, The dosage form of the drug is decoction, pill, capsule, tablet, powder or granule.

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