A phenolic compound, a preparation method thereof, and application thereof in preparing a drug for treating perimenopausal depression

Abstract

The present invention relates to the technical field of natural medicinal chemistry, and specifically to a phenolic compound, a preparation method thereof, and its use in preparing a drug for treating perimenopausal depression. The phenolic compound provided by the present invention has a structure represented by Formula I or Formula II. Data from the examples show that the compounds represented by Formula I and Formula II provided by the present invention have good anti-PMD activity and can be used to prepare a drug for treating perimenopausal depression, filling a technological gap.

Description

Technical Field

[0001] The present invention relates to the technical field of natural medicinal chemistry, and in particular to a phenolic compound, a preparation method thereof, and application of the phenolic compound in preparing a drug for treating perimenopausal depression. Background Art

[0002] Perimenopausal depression (PMD) is a common and highly prevalent mental illness among women worldwide, severely impacting both physical and mental health. PMD differs from conventional depression in that its primary causes are ovarian dysfunction and low estrogen levels. Conventional antidepressants suffer from slow onset of action and poor clinical efficacy against PMD. Hormone replacement therapy (HRT) is also limited in its clinical application due to numerous adverse effects, including the risk of breast cancer. Therefore, the development of new, effective and safe anti-PMD drugs is urgent. While ancient Chinese medical texts do not specifically mention perimenopausal depression, its clinical symptoms share similarities with the TCM concepts of "depression," "visceral agitation," "lily disease," and "plum pit qi." According to TCM, the fundamental pathogenesis of perimenopausal depression is kidney deficiency and liver depression, with kidney deficiency as the root cause and liver depression as the secondary cause. This means that kidney deficiency in women, combined with liver qi stagnation, leads to visceral dysfunction, resulting in imbalances in yin and yang, qi and blood, and mental and spiritual well-being. Therefore, "tonifying the kidney, replenishing essence and nourishing the marrow" is a fundamental principle in TCM treatment of depression.

[0003] Epimedium, a traditional Chinese medicine for kidney tonification, exhibits estrogen-like effects. It not only improves HPA axis dysfunction but also regulates the activity of CYP19, an aromatase key to estrogen synthesis, promoting estrogen synthesis in human ovarian granulosa cells. Indran et al., using a nude mouse model, found that compared with the synthetic estrogen ethinyl estradiol, epimedium extract is estrogen receptor (ER) selective, does not increase the risk of breast cancer, and has a better safety profile. Furthermore, icariin can effectively increase the expression of the hormone receptor ERα in the hypothalamus of rats with PMD and activate the PI3K-AKT signaling pathway mediated by ERα, thereby improving brain neurotransmitter disorders and playing an anti-PMD role. The classic formula Erxian Decoction warms kidney yang, replenishes kidney essence, and regulates the Chong and Ren meridians. It is primarily used to treat perimenopausal syndrome (PMS). It can upregulate ovarian aromatase secretion and enhance estradiol synthesis. Previous research by our group has also shown that Erxian Decoction effectively improves depressive-like behavior in PMD mice, upregulates estradiol levels, and significantly alleviates female endocrine hormone imbalances in these models, increasing the expression of estrogen receptors ERα and ERβ, and boosting BDNF expression in the hippocampus. Miao Mingsan et al. reported that curculigoside effectively improves female endocrine hormone imbalances in mice with perimenopausal depression and increases brain 5-HT and DA levels. Various studies have demonstrated that treating perimenopausal depression through a liver-kidney-mediated approach significantly improves symptoms without increasing the risk of adverse reactions. Therefore, Traditional Chinese Medicine holds great potential for the treatment of PMD.

[0004] Curculigo capitulata is a perennial herbaceous plant of the genus Curculigo in the Curculigaceae family. Its rhizome is used medicinally to warm the yang and tonify the kidneys. The "Compendium of Materia Medica" records that Curculigo capitulata nourishes the kidneys and strengthens essence, treating symptoms such as kidney deficiency, cough and asthma, impotence and spermatorrhea, and uterine coldness and infertility. It is also clearly mentioned in local Chinese medicinal records in Sichuan, Yunnan, and Guangxi Zhuang Autonomous Regions. Curculigo plants primarily contain two characteristic components: simple phenolic compounds, represented by curculigoside and orcinol glucoside, and norlignans, which are based on a diarylpentane (Ph-5C-Ph) skeleton. To date, there are no reports on whether phenolic compounds with pyrrolidin-2-one substituents in Curculigo capitulata exhibit anti-PMD activity. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a phenolic compound and its preparation method and its use in preparing a drug for treating perimenopausal depression. The phenolic compound provided by the present invention has anti-PMD activity, filling a technical gap.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a phenolic compound having a structure shown in Formula I or Formula II:

[0008]

[0009] The present invention provides a method for preparing the phenolic compound described in the above technical solution, comprising the following steps:

[0010] The dried rhizome of Curculigo orchioides and an ethanol aqueous solution are mixed, and the mixture is subjected to reflux extraction and concentration in sequence to obtain an extract;

[0011] Extracting the extract with a weak acid aqueous solution and a weak base aqueous solution in sequence to obtain a weak base extract;

[0012] extracting the weak base extract with ethyl acetate to obtain an ethyl acetate extract;

[0013] The ethyl acetate extract was subjected to a first silica gel column chromatography separation, using a first dichloromethane-methanol system for the first elution, and according to the order of elution of the components, a Fr.2.1 fraction and a Fr.2.2 fraction were obtained;

[0014] The Fr.2.1 fraction was subjected to a second silica gel column chromatography separation, and a second dichloromethane-methanol system was used for a second elution. The resulting silica gel column chromatography eluate was subjected to a first dextran gel column chromatography, and methanol was used for elution. In the order of the elution of the components, three fractions Fr.2.1.1 to Fr.2.1.3 were obtained;

[0015] Sequentially subjecting the Fr.2.1.3 fraction to a second dextran gel column chromatography separation, a medium-pressure preparative chromatography column separation, and a third silica gel column chromatography separation to obtain a phenolic compound having a structure represented by Formula I or Formula II;

[0016] During the second Sephadex column chromatography separation, the eluent is a mixture of dichloromethane and methanol;

[0017] When separating on a medium-pressure preparative column, the eluent is methanol-water solution;

[0018] During the third silica gel column chromatography, the eluent was dichloromethane-acetone system.

[0019] Preferably, the first elution is a gradient elution using a first dichloromethane-methanol system in which the volume ratio of dichloromethane to methanol varies from 50:1 to 10:1.

[0020] Preferably, the second elution is performed by gradient elution using a second dichloromethane-methanol system in which the volume ratio of dichloromethane to methanol changes from 20:1 to 1:1.

[0021] Preferably, the volume ratio of dichloromethane to methanol in the mixed solution of dichloromethane and methanol is 1:1.

[0022] Preferably, the volume ratio of dichloromethane to acetone in the dichloromethane-acetone system is 15:1.

[0023] Preferably, the volume concentration of the methanol aqueous solution is 30%.

[0024] Preferably, the weak acid aqueous solution includes a 3 wt. % hydrochloric acid aqueous solution or a 3 wt. % acetic acid aqueous solution.

[0025] Preferably, the weak base aqueous solution comprises a 3 wt.% sodium hydroxide aqueous solution.

[0026] The present invention also provides the use of the phenolic compound described in the above technical solution or the phenolic compound prepared by the above preparation method in the preparation of a drug for treating perimenopausal depression.

[0027] The present invention provides a phenolic compound having a structure represented by Formula I or Formula II. Data from the examples show that the compound having a structure represented by Formula I and the compound having a structure represented by Formula II provided by the present invention have good anti-PMD activity and can be used to prepare a drug for treating perimenopausal depression, thus filling a technical gap. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the chemical structures of compound 1 and compound 2;

[0029] Figure 2 Comparison of behavioral tests of mice in each group, where: Sham is the sham operation group; Model is the model group; CF (Low) is the low-dose group of compound 1, CF (High) is the high-dose group of compound 2; CG (Low) is the low-dose group of compound 2, CG (High) is the high-dose group of compound 2; Positive is the positive control group. ###P<0.001, ***P<0.001;

[0030] Figure 3 Comparison of serum estrogen levels in mice of each group; ###P<0.001, ***P<0.001;

[0031] Figure 4 Comparison of hippocampal neurotransmitter content in each group of mice; ###P<0.001, ***P<0.001;

[0032] Figure 5 The expression of Erβ, TrKB and BDNF in the hippocampus of mice in each group; ###P<0.001, ***P<0.001. DETAILED DESCRIPTION

[0033] The present invention provides a phenolic compound having a structure shown in Formula I or Formula II:

[0034]

[0035] The present invention also provides a method for preparing the phenolic compound described in the above technical solution, comprising the following steps:

[0036] The dried rhizome of Curculigo orchioides and an ethanol aqueous solution are mixed, and the mixture is subjected to reflux extraction and concentration in sequence to obtain an extract;

[0037] Extracting the extract with a weak acid aqueous solution and a weak base aqueous solution in sequence to obtain a weak base extract;

[0038] extracting the weak base extract with ethyl acetate to obtain an ethyl acetate extract;

[0039] The ethyl acetate extract was subjected to a first silica gel column chromatography separation, using a first dichloromethane-methanol system for the first elution, and according to the order of elution of the components, a Fr.2.1 fraction and a Fr.2.2 fraction were obtained;

[0040] The Fr.2.1 fraction was subjected to a second silica gel column chromatography separation, and a second dichloromethane-methanol system was used for a second elution. The resulting silica gel column chromatography eluate was subjected to a first dextran gel column chromatography, and methanol was used for elution. In the order of the elution of the components, three fractions Fr.2.1.1 to Fr.2.1.3 were obtained;

[0041] Sequentially subjecting the Fr.2.1.3 fraction to a second dextran gel column chromatography separation, a medium-pressure preparative chromatography column separation, and a third silica gel column chromatography separation to obtain a phenolic compound having a structure represented by Formula I or Formula II;

[0042] During the second Sephadex column chromatography separation, the eluent is a mixture of dichloromethane and methanol;

[0043] When separating on a medium-pressure preparative column, the eluent is methanol-water solution;

[0044] During the third silica gel column chromatography, the eluent was dichloromethane-acetone system.

[0045] The invention mixes the dried rhizome of Curculigo macrophylla with an ethanol aqueous solution, and sequentially performs reflux extraction and concentration to obtain an extract.

[0046] As an embodiment, the volume concentration of the ethanol aqueous solution is 95%.

[0047] As an embodiment, the usage ratio of the dried rhizome of Curculigo macrophylla to the ethanol aqueous solution is 6 kg:30 L.

[0048] As an embodiment, the reflux extraction is performed twice, and the time for each extraction is 3 hours.

[0049] After obtaining the extract, the present invention sequentially extracts the extract with a weak acid aqueous solution and a weak base aqueous solution to obtain a weak base extract.

[0050] As an embodiment, the weak acid aqueous solution includes a 3 wt. % hydrochloric acid aqueous solution or a 3 wt. % acetic acid aqueous solution; as an embodiment, the weak base aqueous solution includes a 3 wt. % sodium hydroxide aqueous solution.

[0051] After obtaining the weak base extract, the present invention extracts the weak base extract with ethyl acetate to obtain an ethyl acetate extract.

[0052] As an embodiment, the extraction is performed three times.

[0053] After obtaining the ethyl acetate extract, the present invention performs a first silica gel column chromatography separation on the ethyl acetate extract, adopts a first dichloromethane-methanol system for first elution, and obtains Fr.2.1 fraction and Fr.2.2 fraction according to the elution order of the components.

[0054] As an embodiment, the first elution is a gradient elution using a first dichloromethane-methanol system in which the volume ratio of dichloromethane to methanol varies from 50:1 to 10:1.

[0055] After obtaining the Fr.2.1 fraction, the present invention subjects the Fr.2.1 fraction to a second silica gel column chromatography separation, employing a second dichloromethane-methanol system for a second elution, and subjecting the resulting silica gel column chromatography eluate to a first dextran gel column chromatography, employing methanol for elution, to obtain three fractions, Fr.2.1.1 to Fr.2.1.3, in the order of component elution.

[0056] As an embodiment, the second elution is performed by gradient elution using a second dichloromethane-methanol system in which the volume ratio of dichloromethane to methanol changes from 20:1 to 1:1.

[0057] After obtaining the Fr.2.1.3 fraction, the present invention sequentially subjects the Fr.2.1.3 fraction to a second dextran gel column chromatography separation, a medium-pressure preparative chromatography column separation, and a third silica gel column chromatography to obtain a phenolic compound having a structure represented by Formula I or Formula II.

[0058] As an embodiment, during the second dextran gel column chromatography separation, the eluent is a mixture of dichloromethane and methanol; the volume ratio of dichloromethane to methanol in the mixture of dichloromethane and methanol is 1:1.

[0059] As an embodiment, during separation using a medium-pressure preparative chromatography column, the eluent is a methanol-water solution, and the volume concentration of the methanol-water solution is 30%.

[0060] As an embodiment, during the third silica gel column chromatography, the eluent is a dichloromethane-acetone system, and the volume ratio of dichloromethane to acetone in the dichloromethane-acetone system is 15:1.

[0061] The preparation method of the compound having the structure represented by Formula I and Formula II provided by the present invention is simple, easy to implement, environmentally friendly and material-saving.

[0062] The present invention also provides the use of the phenolic compound described in the above technical solution or the phenolic compound prepared by the preparation method described in the above technical solution in the preparation of a drug for treating perimenopausal depression.

[0063] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0064] Example 1

[0065] The dried rhizome of Curculigo macrophylla (about 6 kg) was extracted twice with 30 L of 95 vol.% ethanol aqueous solution at 60°C in a multifunctional vacuum extraction and concentration tank under reflux, each time for 3 hours. The combined extracts were distilled under reduced pressure until no extract remained, to obtain about 1250 g of Curculigo macrophylla ethanol extract.

[0066] The obtained extract was extracted with 1 L of a 3 wt.% hydrochloric acid aqueous solution, and the extract was mixed with 1 L of a 3 wt.% sodium hydroxide aqueous solution, followed by extraction three times with ethyl acetate. The extract was concentrated to obtain 36 g of an extract.

[0067] 36 g of the ethyl acetate extract was separated by silica gel column chromatography and eluted with a dichloromethane-methanol system (the volume ratio of dichloromethane to methanol was changed from 50:1→1:1) to obtain Fr.2.1 fraction and Fr.2.2 fraction.

[0068] 10gFr.2.1 fraction was separated by silica gel column chromatography and eluted with a dichloromethane-methanol system (the volume ratio of dichloromethane and methanol changed from 20:1→1:1) to obtain an eluate; the obtained eluate was separated by Sephadex LH-20 column chromatography and eluted with methanol to obtain Fr.2.1.1 fraction, Fr.2.1.2 fraction and Fr.2.1.3 fraction.

[0069] The 1.5 g Fr.2.1.3 fraction was separated by Sephadex LH-20 column chromatography and eluted with a mixture of dichloromethane and methanol (the volume ratio of dichloromethane to methanol was 1:1). The eluate was separated by medium-pressure preparative chromatography (ODS column separation, eluted with 30 vol.% methanol aqueous solution, and the eluate was separated by silica gel column chromatography and eluted with a dichloromethane-acetone system (the volume ratio of dichloromethane to acetone was 15:1) to obtain 410 mg of compound 1 with the structure shown in Formula I and 380 mg of compound 2 with the structure shown in Formula II.

[0070] 1. Detection:

[0071] The present invention performs ultraviolet spectrum detection, infrared spectrum detection, nuclear magnetic resonance hydrogen spectrum detection, carbon spectrum detection and high-resolution mass spectrum detection on compound 1 and compound 2.

[0072] The data characterization of compound 1 was as follows: amorphous powder; (c 0.1,MeOH); UV(MeOH)λ max (logε)208(3.09),284(2.46)nm; IRν max :3393.0,2678.1,1646.5,1593.2,1427.0,1372.7,1280.6,1160.7,1060.9,984.9,824.5,641.0,589.0,514.8cm -1 ; 1 H (600 MHz, MeOH), 13 C (175 MHz, MeOH) NMR data, see Table 1; (+)-HRESIMS m / z 230.0826 [M+Na] + (calcd.forC 11 H 13 NNaO3230.0793).

[0073] The data characterization of compound 2 is: amorphous powder; (c 0.1,MeOH); UV(MeOH)λ max (logε)208(3.09),284(2.46)nm; IRν max :3393.0,2678.1,1646.5,1593.2,1427.0,1372.7,1280.6,1160.7,1060.9,984.9,824.5,641.0,589.0,514.8cm -1 ; 1 H (600 MHz, MeOH), 13C (175 MHz, MeOH) NMR data, see Table 1; (+)-HRESIMS m / z 230.0826 [M+Na] + (calcd.forC 11 H 13 NNaO3230.0793).

[0074] Table 1 H NMR and C NMR data of compound 1 and compound 2 in CD3OD

[0075]

[0076]

[0077] 2. Therapeutic effects of compounds 1 and 2 on PMD model mice

[0078] Experimental animals

[0079] SPF female ICR mice; 8 weeks old; weight 20-25 g; provided by: Shanghai Slake Laboratory Animal Co., Ltd.; Animal Ethics Certificate Number: LLSC20220879.

[0080] 2.2. Preparation of the bilateral ovariectomized (OVX) model

[0081] Mice were anesthetized with isoflurane, fixed in a supine position, and the skin was prepared. The skin from the lowest rib margin to the sacrum of the rat's back was thoroughly disinfected with iodine tincture, and then a longitudinal incision with a long diameter of about 2 cm was made. The skin and fascia were cut layer by layer, the lumbar muscle layer was opened, and the adipose tissue was found and pulled out. The mouse cauliflower-like ovary was found in it. The ovary was removed after ligation along the distal end of the cervix to stop bleeding. After the operation, the remaining tissue was returned to the abdominal cavity, the muscles and skin were sutured and thoroughly disinfected with iodine tincture, and the same operation was performed on both sides. Penicillin was injected intramuscularly at a dose of 200,000 U / mouse to prevent infection. The sham operation group opened the abdominal cavity after anesthesia, found the ovary but did not remove it, and only removed the fat mass of the same size near it and sutured it in the same way. Other surgical procedures were the same as those of the surgical group.

[0082] The mice were randomly divided into 7 groups, with 8 mice in each group: sham operation group, model group, positive control group, low-dose and high-dose compound 1 groups, and low-dose and high-dose compound 2 groups.

[0083] 2.3. Establishment of the Chronic Unpredictable Mild Stress (CUMS) Model

[0084] On the eighth day after surgery, rats in each group underwent CUMS modeling. All mice, except the sham-operated group, were housed individually underwent CUMS stimulation. The stimulation consisted of 24-hour food and water deprivation, 24-hour moist bedding, 12-hour cage tilting, 5-minute ice-water swimming, 15-minute cage shaking, 1-hour ultrasound stimulation, and a light-dark reversal. Each stimulus was randomly selected daily to prevent the mice from anticipating it and developing habituation. Each stimulus was applied an average of 2–3 times, and the same stimulus was not repeated in the same mouse within two days for a total of 21 days.

[0085] Mice in the low-dose compound 1 group (8.65 mg / kg), high-dose compound 1 group (70 mg / kg), low-dose compound 2 group (8.65 mg / kg) and high-dose compound 2 group (70 mg / kg) were gavaged with the corresponding dose of compound 1 hour before CUMS intervention, once a day; mice in the sham operation group and model group were given an equal volume (1 mL / 100 g) of 0.9% NaCl solution; mice in the positive control group were given 0.21 g / mL fluoxetine hydrochloride and estradiol suspension, and all were administered continuously for 21 days.

[0086] 2.4. Evaluation of depressive behavior

[0087] Behavioral assessment was performed after 21 days of treatment.

[0088] 2.4.1. Forced swim test (FST)

[0089] Mice were placed in a 50 cm high, 20 cm diameter cylindrical transparent container. Each mouse was tested for 6 minutes, with the first 2 minutes being the acclimatization period. The duration of the mouse's buoyancy at the water surface (minor movements to maintain oral and nasal breathing were permitted) was recorded for the next 4 minutes. The water was changed after each test.

[0090] 2.4.2. Tail suspension test (TST)

[0091] Tie one end of the suspension string 2 cm from the mouse's tail. Suspend the mouse in mid-air (approximately 50 cm above the ground) and secure it with tape to prevent it from falling due to struggling during the experiment. After acclimating for 1 minute, measure the duration of immobility in the suspended state for 5 minutes.

[0092] 2.4.3. Open field test (OFT)

[0093] A black-walled open-field chamber (80 cm × 80 cm × 40 cm) with a bottom composed of 25 equal-sized blocks was used. A video camera was placed directly above the chamber. During the experiment, mice were placed in the central grid at the bottom of the chamber, and their movements were recorded for 3 minutes. After each test, the chamber was wiped with 75% ethanol to prevent the "predecessor effect." Videos were analyzed using SuperMaze V2.0 software, with the total distance traveled (path length) as the evaluation metric.

[0094] 2.4.4 Behavioral measurement results

[0095] The immobility time of mice in the model group in the forced swimming and tail suspension tests was significantly longer than that in the sham operation group ( Figure 2 AB, ##P<0.01, ###P<0.001, ***P<0.001), indicating that the model was successfully established; the immobility time of mice in the high-dose and low-dose groups of compound 1, high-dose and low-dose groups of compound 2, and the positive control group in the forced swimming and tail suspension tests was shortened to varying degrees. In the open field test, the path length of mice in the model group was significantly shorter than that in the sham operation group, and the path length of mice in the high-dose and low-dose groups of compound 1, high-dose and low-dose groups of compound 2, and the positive control group was significantly increased compared with the model group ( Figure 2 (C) The results of forced swimming, tail suspension and open field tests showed that compounds 1 and 2 had an ameliorative effect on PMD.

[0096] Determination of hormone levels in serum of 3.5PMD mice

[0097] After the behavioral experiment, blood was collected from the rats' orbits. The whole blood was allowed to rest at room temperature for 4 hours and then centrifuged (at 3000 rpm) for 15 minutes. The supernatant was collected as serum, aliquoted, and stored at -80°C until use. It was thawed at 4°C before use. The levels of estradiol (E2), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) in the serum of the mice in each group were measured using enzyme-linked immunosorbent assay (ELISA). The assay procedures were carried out according to the kit instructions.

[0098] Compared with the normal control group, the serum E2 content of mice in the model control group was significantly decreased (P<0.001), and the LH and FSH contents were significantly increased; compared with the model, the serum E2 content of mice in the high-dose and low-dose groups of compound 1, the high-dose and low-dose groups of compound 2, and the positive control group was significantly increased (P<0.001), and the LH and FSH contents were significantly decreased. Figure 3 .

[0099] 2.6. Determination of neurotransmitter levels in serum of PMD mice

[0100] The levels of dopamine (DA), norepinephrine (NE), and 5-hydroxytryptamine (5-HT) in the serum obtained in 2.5 were detected by ELISA. Compared with the normal control group, the serum DA, NE, and 5-HT levels of the mice in the model control group were significantly reduced (P<0.001); compared with the model, the serum DA, NE, and 5-HT levels of the mice in the high- and low-dose groups of compound 1, the high- and low-dose groups of compound 2, and the positive control group were significantly increased (P<0.001). Figure 4 .

[0101] 2.7. Study on the anti-PMD mechanism of action of compounds

[0102] 2.7.1. Tissue protein extraction

[0103] After the behavioral experiments, mice in each group were sacrificed by cervical dislocation. Hippocampal tissue was rapidly isolated on ice, snap-frozen in liquid nitrogen, and stored at −80°C until use. Tissue blocks were added to an appropriate volume of RIPA lysis buffer and homogenized using an automated grinder. The supernatant was centrifuged at 12,000 rpm for 10 minutes at 4°C, and the protein concentration was measured using a BCA assay. The supernatant was diluted to 1× using 5× Loading Buffer, vortexed, and incubated at 100°C in a metal bath for 5 minutes before storage at −20°C.

[0104] 2.7.2.Western Blot

[0105] Prepare a 10% SDS-PAGE gel separating and stacking gel, load the sample, and run electrophoresis at a constant voltage of 80 V for approximately 30 minutes. Once the sample enters the separating gel along with the bromophenol blue, adjust the voltage to 120 V. The run time is determined based on the protein's molecular weight. After the run, cut the gel to the desired protein range, referring to the molecular weight indicated by the marker. Transfer the protein to a PVDF membrane at a constant current of 250 mA. After transfer, block the membrane with 5% skim milk powder (prepared with TBS buffer containing 0.1% Tween-20) in TBST for 1 hour at room temperature. Incubate the PVDF membrane with the primary antibody overnight at 4°C. The next day, wash the membrane three times with TBST, incubate with the secondary antibody (1:9000) at room temperature for 1 hour, and then wash the membrane three times with TBST. Cover the PVDF membrane with ECL luminescence solution, then develop and capture images on a molecular gel imaging system. Bands are analyzed using Image Lab 5.1 software. The expression level of the target protein is expressed as the grayscale ratio relative to the internal control GAPDH.

[0106] 2.7.3 Experimental results

[0107] The results showed that compared with the sham operation group, the expression of TrKB, BDNF and Erα in the Model group was significantly decreased (p<0.05 or p<0.01). After drug intervention, the high-dose and low-dose groups of compound 1, the high-dose and low-dose groups of compound 2 and the positive control group could significantly upregulate the expression of TrkB, BDNF and ERβ (p<0.05). Figure 5 .

[0108] From the above experimental results, it can be seen that compounds 1 and 2 significantly improved OVX-CUMS-induced depressive-like behavior by regulating the expression of TrKB, BDNF, and Erβ in the hippocampus, increasing serum estradiol, reducing serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH), and promoting the release of neurotransmitters DA, NE, and 5-HT.

[0109] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for preparing a phenolic compound, characterized in that: The following steps are involved: The dried rhizome of Curculigo orchioides and an ethanol aqueous solution are mixed, and the mixture is subjected to reflux extraction and concentration in sequence to obtain an extract; Extracting the extract with a weak acid aqueous solution and a weak base aqueous solution in sequence to obtain a weak base extract; extracting the weak base extract with ethyl acetate to obtain an ethyl acetate extract; The ethyl acetate extract was subjected to a first silica gel column chromatography separation, using a first dichloromethane-methanol system for the first elution, and according to the order of elution of the components, a Fr.2.1 fraction and a Fr.2.2 fraction were obtained; The Fr.2.1 fraction was subjected to a second silica gel column chromatography separation, and a second dichloromethane-methanol system was used for a second elution. The resulting silica gel column chromatography eluate was subjected to a first dextran gel column chromatography, and methanol was used for elution. In the order of the elution of the components, three fractions Fr.2.1.1 to Fr.2.1.3 were obtained; Sequentially subjecting the Fr.2.1.3 fraction to a second dextran gel column chromatography separation, a medium-pressure preparative chromatography column separation, and a third silica gel column chromatography separation to obtain a phenolic compound having a structure represented by Formula I or Formula II; During the second Sephadex column chromatography separation, the eluent is a mixture of dichloromethane and methanol; When separating on a medium-pressure preparative column, the eluent is methanol-water solution; During the third silica gel column chromatography, the eluent was dichloromethane-acetone system.

2. The preparation method according to claim 1, characterized in that The first elution is a gradient elution using a first dichloromethane-methanol system with a volume ratio of dichloromethane to methanol varying from 50:1 to 10:

1.

3. The preparation method according to claim 1, characterized in that The second elution is performed by gradient elution using a second dichloromethane-methanol system with a volume ratio of dichloromethane to methanol varying from 20:1 to 1:

1.

4. The preparation method according to claim 1, characterized in that The volume ratio of dichloromethane to methanol in the mixed solution of dichloromethane and methanol is 1:

1.

5. The preparation method according to claim 1, wherein The volume ratio of dichloromethane to acetone in the dichloromethane-acetone system is 15:

1.

6. The preparation method according to claim 1, wherein The volume concentration of the methanol aqueous solution is 30%.

7. The preparation method according to claim 1, wherein The weak acid aqueous solution is a 3 wt. % hydrochloric acid aqueous solution or a 3 wt. % acetic acid aqueous solution.

8. The preparation method according to claim 1, wherein The weak base aqueous solution is a 3 wt. % sodium hydroxide aqueous solution.

9. Use of the phenolic compound prepared by the preparation method according to any one of claims 2 to 8 in the preparation of a medicament for treating perimenopausal depression.

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