Hyperforidiol E, a phloroglucinol compound, its preparation method and application

Abstract

The application belongs to the technical field of medicines, and specifically discloses a m-benzenetriol compound Hyperforidiol E, a preparation method and application thereof. Experimental results show that the m-benzenetriol compound Hyperforidiol E provided by the application has significant antidepressant activity, is superior to positive drugs fluoxetine and Luoyoutei, and can be used for developing drugs for treating depression.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and more specifically, relates to a phloroglucinol compound Hyperforidiol E, its preparation method, pharmaceutical composition, and its application in the treatment of antidepressants. Background Technology

[0002] St. John's wort is a perennial herb belonging to the genus Hypericum in the family Clusiaceae. The dried aerial parts are used medicinally. It has a pungent and cold nature and enters the liver meridian. It grows on hillsides, roadsides, grasslands, under forests, and riverbanks. It is widely distributed in Southern Europe, Cyprus, Northwest Africa, the Near East, Central Asia, India to Mongolia, and in Hebei, Shanxi, Shaanxi, Gansu, Xinjiang and other places in my country.

[0003] In search of new drugs for treating depression, the inventors isolated a novel phloroglucinol compound, Hyperforidiol E, from the dried aerial parts of St. John's wort and found that it has significant antidepressant activity, which is significantly better than the positive control drugs fluoxetine and Neurostan, and can be used to develop antidepressant drugs. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a novel class of phloroglucinol compounds with 2-hydroxypropyl-epoxyhexane substitutions at positions 2 and 8, along with their preparation methods, pharmaceutical compositions, and applications.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] A novel phloroglucinol compound, Hyperforidiol E, has the structure shown in formula (1):

[0007]

[0008] Tautomers of the novel phloroglucinol compound Hyperforidiol E or pharmaceutically acceptable salts thereof are also within the scope of protection of this invention.

[0009] In another aspect, the present invention also provides a method for preparing the above-mentioned novel phloroglucinol compound Hyperforidiol E, wherein the novel phloroglucinol compound Hyperforidiol E is extracted and isolated from St. John's wort.

[0010] In the above technical solution, the preparation method of the novel phloroglucinol compound Hyperforidiol E includes the following steps:

[0011] S1. Take the dried aerial parts of St. John's wort, add solvent and reflux to extract, combine the extracts and concentrate to obtain the extract.

[0012] S2. After suspending the extract in water, extract it with petroleum ether to obtain the extract;

[0013] S3. The extract was subjected to gradient elution using a petroleum ether-ethyl acetate solution via silica gel column chromatography to obtain fraction E. When identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction E was 0.65-0.68.

[0014] S4. Fraction E was subjected to gradient elution using petroleum ether-ethyl acetate solution via silica gel column chromatography to obtain fraction E2; when identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction E2 was 0.75-0.78.

[0015] S5. Fraction E2 was subjected to gradient elution using methanol-water solution via MCI column chromatography to obtain fraction E2B; when identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction E2B was 0.47-0.49.

[0016] S6. Hyperforidiol E, a phloroglucinol compound having the structure shown in formula (1), was obtained by separation from fraction E2B using HPLC.

[0017] The mobile phase used in the HPLC method is acetonitrile-water solution.

[0018] This application uses acetonitrile-water as the mobile phase and employs HPLC method, utilizing C 18 Hyperforidiol E, a phloroglucinol compound with the structural formula shown in formula (1), was prepared by chromatographic column in fraction E2B.

[0019] Further, in step S6, the volume ratio of acetonitrile to water in the acetonitrile-water solution is (55:45)-(65:35). Preferably, the volume ratio of acetonitrile to water in the acetonitrile-water solution is 62:38.

[0020] Further, in step S6, the characteristic ultraviolet absorption (λ) of phloroglucinol observed by HPLC analysis is... max Further separation of fraction E2B was performed at 220, 280 nm, and the retention time of the obtained phloroglucinol compounds was 50-60 min.

[0021] Further, in step S2, the extract is suspended in 8-15 times its weight of water and then extracted with petroleum ether.

[0022] In this application, the Rf value is the Rf value of the fluorescent spot observed at scanning wavelengths of 254 nm and 365 nm.

[0023] Further, in step S3, based on a total volume of 100, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution when washing out fraction E (i.e., the fraction corresponding to E) is (45:55)-(55:45); preferably, the volume ratio of petroleum ether to ethyl acetate is 50:50.

[0024] Further, in step S4, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution when washing out fraction E2 (i.e., the fraction corresponding to E2) is (75:25)-(85:15); preferably, the volume ratio of petroleum ether to ethyl acetate is 80:20.

[0025] Further, in step S5, the volume ratio of methanol to water in the methanol-water solution when washing out fraction E2B (i.e., the volume ratio of methanol to water corresponding to fraction E2B) is (65:35)-(72:28); preferably, the volume ratio of methanol to water is 70:30.

[0026] Further, in step S1, the solvent is an 88-98% aqueous ethanol solution; the solvent added is 8-10 times the mass of St. John's wort, and the reflux extraction is performed 1-3 times, with each extraction lasting 1-3 hours.

[0027] In another aspect, the present invention provides a pharmaceutical composition containing the novel phloroglucinol compound Hyperforidiol E described above.

[0028] Furthermore, the pharmaceutical composition includes an synergist and a pharmacodynamically acceptable carrier or excipient.

[0029] In other words, pharmaceutical compositions containing the phloroglucinol compounds of the present invention as active ingredients and conventional pharmaceutical excipients, adjuvants or carriers are also included in the present invention.

[0030] Furthermore, the synergist is one or more of the following substances:

[0031] Fluoxetine, paroxetine, fluvoxamine, sertraline, citalopram, escitalopram, venlafaxine, duloxetine, mirtazapine, bupropion, agomelatine, trazodone, reboxetine, imipramine, amitriptyline, clomipramine, doxepin, maprotiline, moclobemide, liver-soothing and mood-regulating capsules, St. John's wort extract, flupentixol melitracen.

[0032] Furthermore, the dosage form of the pharmaceutical composition is tablets, capsules, granules, oral liquids, powders, drops, or micro-pellets.

[0033] In another aspect, the present invention provides the use of the above-mentioned novel phloroglucinol compound Hyperforidiol E or the above-mentioned composition in antidepressant drugs.

[0034] Compared with the prior art, the present invention has the following advantages:

[0035] (1) This invention provides a novel phloroglucinol compound, Hyperforidiol E, which has not been reported before. Further research has found that the compound Hyperforidiol E has a significant protective effect on corticosterone-induced SH-SY5Y cells, which is superior to positive control drugs fluoxetine and loratadine. It can be used to develop drugs for the treatment of depression.

[0036] (2) The preparation method of the novel phloroglucinol compound Hyperforidiol E is simple, reproducible and has high extraction purity. Attached Figure Description

[0037] Figure 1 The Hyperforidiol E prepared in Example 1 of this invention 1 1H NMR spectrum (400MHz, CDCl3);

[0038] Figure 2 The Hyperforidiol E prepared in Example 1 of this invention 13 C10 NMR spectrum (100MHz, CDCl3);

[0039] Figure 3 The image shows the HMBC spectrum of Hyperforidiol E obtained in Example 1 of this invention. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments.

[0041] It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0042] In the embodiments, unless otherwise specified, all methods used are conventional methods in the art.

[0043] The terms “comprising,” “including,” or any other variations thereof, as used herein, are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0044] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0045] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0046] Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0047] Example 1

[0048] This embodiment discloses a method for preparing a novel phloroglucinol compound, comprising the following steps:

[0049] S1. Take the dried aerial parts of St. John's wort, add solvent and reflux extract, combine the extracts and concentrate to obtain an extract; specifically, the solvent is an 88-98% ethanol aqueous solution; the mass of the solvent added is 8-10 times that of St. John's wort, the number of reflux extractions is 1-3, and each extraction is 1-3 hours.

[0050] A specific implementation plan is as follows: Weigh 208 kg of dried aerial parts of St. John's wort, add 10 times the amount of 95% ethanol aqueous solution as solvent and reflux extract three times, each time for 2 hours. Combine the extracts and concentrate to obtain an extract (about 9.0 kg).

[0051] S2. After suspending the extract in water, extract it with petroleum ether to obtain the extract;

[0052] Further, in step S2, the extract is suspended in 8-15 times its weight of water and then extracted with petroleum ether.

[0053] The specific steps are as follows: After suspending the extract (9.0 kg) in 10 times its weight of water (90 L), extract it with 1.5 times its volume of petroleum ether, and extract it 3 times to obtain petroleum ether extract (about 3100 g).

[0054] S3. The extract is subjected to gradient elution using a petroleum ether-ethyl acetate solution via silica gel column chromatography to obtain fraction E. When identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction E is 0.65-0.68. Based on a total volume of 100, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution at the time of elution of fraction E (i.e., the volume ratio corresponding to fraction E) is (45:55)-(55:45). Preferably, the volume ratio of petroleum ether to ethyl acetate is 50:50.

[0055] It should be noted that during gradient elution, petroleum ether-ethyl acetate solutions with a volume ratio of (45:55) to (55:45) can all elute fraction E. Fraction E is identified by silica gel thin-layer chromatography with an Rf value of 0.65-0.68. The elution efficiency is highest for a petroleum ether-ethyl acetate solution with a volume ratio of 50:50. The "preferred" principle described in the following gradient elution processes is the same.

[0056] In this application, the Rf value is the Rf value of the fluorescent spot observed at scanning wavelengths of 254 nm and 365 nm.

[0057] The specific steps are as follows: The petroleum ether extract was subjected to silica gel column chromatography, using a petroleum ether-ethyl acetate gradient elution with volume ratios of 100:0, 90:10, 80:20, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, with each 500 mL volume representing one fraction. A total of 56 fractions were obtained: 1-5, 6-10, 11-25, 26-29, 30-40, 41-44, 45-47, 48-51, 52-53, and 54-56. These fractions were identified using silica gel thin-layer chromatography. The Rf values ​​observed at 254 nm and 365 nm were 0.81-0.83 (fractions 1-5) and 0. Fluorescent spot results of 78-0.80 (fractions 6-10), 0.75-0.77 (fractions 11-25), 0.72-0.74 (fractions 26-29), 0.65-0.68 (fractions 30-40), 0.64-0.63 (fractions 41-44), 0.57-0.62 (fractions 45-47), 0.52-0.54 (fractions 48-51), 0.46-0.47 (fractions 52-53), and 0.35-0.39 (fractions 54-56) were combined to obtain 10 fractions, which were named A, B, C, D, E, F, G, H, I, and J, respectively; among them, fraction E was obtained by combining fractions 30-40.

[0058] The characteristic ultraviolet absorption (λ) of phloroglucinol observed by HPLC analysis max =220, 280nm) Select fraction E for the next separation step.

[0059] S4. Fraction E is eluted by gradient chromatography using a petroleum ether-ethyl acetate solution via silica gel column chromatography to obtain fraction E2. When identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction E2 is 0.75-0.78. The volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution used to elute fraction E2 (i.e., the solution corresponding to fraction E2) is (75:25)-(85:15). Preferably, the volume ratio of petroleum ether to ethyl acetate is 80:20.

[0060] The specific operating steps include: passing fraction E through silica gel column chromatography, using a petroleum ether-ethyl acetate gradient elution with volume ratios of 100:0, 80:20, 60:40, 50:50, 30:70, 20:80, 10:90, and 0:100 to sequentially obtain 115 fractions: 1-14, 15-35, 36-38, 39-55, 56-77, 78-80, 81-100, and 101-115. These fractions are then identified using silica gel thin-layer chromatography, and their classification is based on the Rf values ​​of the fluorescent spots observed at wavelengths of 254 nm and 365 nm. The fractions 0.79-0.85 (fractions 1-14), 0.75-0.78 (fractions 15-35), 0.71-0.74 (fractions 36-38), 0.63-0.69 (fractions 39-55), 0.50-0.62 (fractions 56-77), 0.45-0.49 (fractions 78-80), 0.35-0.44 (fractions 81-100), and 0.30-0.34 (fractions 101-115) are combined to obtain fractions E1 to E8; among them, fractions 15-35 are combined to form fraction E2.

[0061] S5. Fraction E2 is eluted by gradient elution using a methanol-water solution via MCI column chromatography to obtain fraction E2B. When identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction E2B is 0.47-0.49. The volume ratio of methanol to water in the methanol-water solution eluted from fraction E2B (i.e., the volume ratio corresponding to fraction E2B) is (65:35)-(72:28). Preferably, the volume ratio of methanol to water is 70:30.

[0062] The specific procedures include: analyzing the characteristic ultraviolet absorption (λ) of phloroglucinol observed by HPLC. max =220, 280nm) Selected E2 for further separation. Fraction E2 was subjected to MCI column chromatography, using methanol-water gradient elution with volume ratios of 60:40, 70:30, 75:25, 80:20, and 90:10, and 35 fractions (1-8, 9-17, 18-23, 24-30, and 31-35) were collected sequentially. Silica gel thin-layer chromatography was used for identification. Based on the Rf values ​​of the fluorescent spots observed at wavelengths of 254nm and 365nm, which were 0.31-0.34 (fractions 1-8), 0.47-0.49 (fractions 9-17), 0.55-0.62 (fractions 18-23), 0.64-0.69 (fractions 24-30), and 0.72-0.75 (fractions 31-35), respectively, five fractions were combined to obtain five fractions, E2A to E2E. Among them, fractions 9-17 are combined to form fraction E2B.

[0063] S6. Hyperforidiol E, a phloroglucinol compound with the structure shown in formula (1), was isolated from fraction E2B by HPLC. The mobile phase used in the HPLC method was acetonitrile-water solution. The volume ratio of acetonitrile to water in the acetonitrile-water solution was (55:45)-(65:35).

[0064] The characteristic ultraviolet absorption (λ) of phloroglucinol observed by HPLC analysis max =220, 280 nm) Select fraction E2B for further separation; The retention time of the obtained novel phloroglucinol compound is 50-60 min;

[0065] More preferably, the volume ratio of acetonitrile to water in the acetonitrile-water solution is 62:38; the retention time of the compound Hyperforidiol E prepared in fraction E2B is 55 min (purity 98%).

[0066] In this application, the Rf value is the Rf value of the fluorescent spot observed at scanning wavelengths of 254 nm and 365 nm.

[0067] The physical properties and detection data of compound 1 obtained in Example 1 are as follows:

[0068] A colorless, oily substance, readily soluble in chloroform and methanol. Based on high-resolution mass spectrometry, its molecular weight is estimated to be 586, and its molecular formula is determined to be C2. 35 H 54 O7 has an unsaturation degree of 9.

[0069] like Figure 1 As shown, 1 δ in H-NMR (CDCl3, 400MHz) spectrum H 5.02 (2H, m) indicates the presence of two double-bonded protons connected to the methylene group in the structure. Furthermore, δ H 3.81 (1H, d, J = 9.4 Hz) suggests a hydroxymethyl group signal; δ H 3.21 (1H, dd, J = 9.6, 2.0 Hz) suggests a hydroxymethyl group linked to a methylene group; δ H 3.28 (1H, dd, J = 15.1, 6.2 Hz) and 3.16 (1H, dd, J = 15.1, 6.0 Hz) suggest a group of methylene signals linked to olefin bonds, and 10 groups of methyl hydrogen signals [δ]. H1.70(3H,s), 1.68(3H,s), 1.67(3H,s), 1.65(3H,s), 1.24(3H,s), 1.21(3H,s), 1.1 7(3H,s), 1.17(3H,d,J=6.2Hz), 1.12(3H,s), 1.10(3H,d,J=6.6Hz), 0.98(3H,s)].

[0070] like Figure 2 As shown, 13 The C-NMR (CDCl3, 100MHz) spectrum showed a total of 35 carbon signals, including 3 carbonyl carbon signals (δ¹⁰). C 211.5, 206.4, 194.3), 1 hydroxyl group carbon signal δ C 165.7, 5 olefin carbon signals (δ) C 134.3, 133.6, 129.9, 122.0, 119.5), one quaternary carbon signal δ C 45.7, 6 methylene carbon signals (δ C 42.3, 33.8, 30.7, 29.5, 24.1, 22.8), 2 methylene carbon signals (δ C 41.1, 35.0), 10 methyl carbon signals (δ C 26.4, 26.1, 26.0, 25.6, 25.5, 23.2, 21.5, 20.7, 18.1, 18.1, 16.7). In addition, it includes two oxygen-linked tertiary carbon signals (δ). C 87.8, 79.0); 2 oxygen-linked quaternary carbon signals (δ C 73.3, 72.6); Quaternary carbon signals of two carbonyl groups (δ C 74.5, 64.4).

[0071] 1 H NMR, 13 The signal assignments for C NMR are shown in Table 1.

[0072] Table 1. NMR data δ (ppm) for compound 1

[0073]

[0074] like Figure 3 The HMBC spectrum shows that H-11(δ) H 2.50) and C-13 (δ C 20.7), C-10(δ) C The correlation between 211.7) indicates the presence of an isobutyryl group in the compound; furthermore, H-27 (δ) H 5.02) and C-26 (δ C22.8), C-3(δ) C 129.9), C-29 (δ) C 25.5), H-31 (δ) H 29.5) and C-4(δ) C 194.3), C-3(δ) C The long-range correlation between H-22(δ) and H-22(δ) demonstrates that the two 3-methyl-2-enbutyl groups are attached to the carbons at the 3 and 5 positions, respectively. H 3.21) and C-21(δ) C 30.7), C-7 (δ) C 35.0), C-23 (δ) C 73.3), C-24 (δ) C 26.4), C-25 (δ) C The relationship between 23.2) indicates that 2,3-dihydroxy-3-methylbutyl is attached to the 7th carbon. Comparison with the known compound Uralione L reveals that compound 1 has a different configuration at the 22nd carbon, i.e., compound 1 is in the R configuration.

[0075] In summary, the structure of the new compound 1 (compound Hyperforidiol E) was determined as follows:

[0076]

[0077] Comparative Example 1

[0078] Comparative Example 1 of the present invention provides a method for preparing a novel phloroglucinol compound. The steps are similar to those in Example 1, except that in step S5, when fraction E2 is subjected to MCI column chromatography, the methanol-water mixture with a methanol-water volume ratio of less than (65:35) or greater than (75:25) is used for elution.

[0079] The results showed that compound 1 could not be prepared.

[0080] Comparative Example 2

[0081] Comparative Example 2 of the present invention provides a method for preparing a novel phloroglucinol compound, the steps of which are similar to those of Example 1, except that in step S6, the volume ratio of acetonitrile to water in the mobile phase used for separating fraction E2B by HPLC is less than 55:45 or greater than 65:35.

[0082] The results showed that compound 1 could not be prepared.

[0083] To better understand the essence of this invention, the following describes the new applications of the above-mentioned phloroglucinol compounds in the pharmaceutical field in conjunction with pharmacological tests and results.

[0084] Test case

[0085] This experimental example discloses the antidepressant activity of the aforementioned compound Hyperforidiol E in corticosterone-induced SH-SY5Y cells (a commonly used cell model for antidepressants).

[0086] (1) Experimental materials and instruments

[0087] Human neuroblastoma SH-SY5Y was purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.

[0088] DMEM medium, PBS buffer, fetal bovine serum (FBS) and trypsin were all purchased from Gibco, USA.

[0089] Fluoxetine (Shanghai Yuanye Biotechnology Co., Ltd.);

[0090] Luyutai (Dr. Wima Schuppe Pharmaceuticals, Germany);

[0091] CCK-8 Cell Viability Assay Kit (Elabscience);

[0092] 96-well plate (Corning Corporation, USA);

[0093] Carbon dioxide cell incubator (Thermo Fisher Scientific, USA);

[0094] Full-wavelength microplate reader (Thermo Fisher Scientific, USA);

[0095] BIOFUGE STRATOS centrifuge (Thermo Fisher Scientific, USA);

[0096] IX73 inverted electron microscope (Olympus Corporation, Japan);

[0097] Pipettes (Eppendorf, Germany);

[0098] Electronic balance (Sartorius GmbH, Germany);

[0099] Clean benches, centrifuge tubes, straws and other related consumables.

[0100] (2) Establishment and evaluation of a corticosterone-damaged SH-SY5Y cell model

[0101] Remove the labeled SH-SY5Y cell cryopreservation tubes from liquid nitrogen and immediately place them in a 37°C water bath to thaw them rapidly within 1 minute. After disinfecting the cryopreservation tubes with alcohol, transfer the cryopreservation solution into a 15mL sterile centrifuge tube, add the appropriate culture medium, mix well, and centrifuge to remove the supernatant. Repeat the above steps once for washing, then add 10mL of culture medium, mix well, resuspend the cells, transfer them to a 10mL culture dish, and incubate in a 37°C, 5% CO2 incubator.

[0102] When the cell density reaches 70%-80%, passage the cells. First, remove the old culture medium, wash twice with PBS, add trypsin containing EDTA to the culture dish and incubate at 37°C for 3 minutes. Then, add culture medium to stop digestion, centrifuge at 1000 rpm for 5 minutes, remove the digestion solution, add serum-containing culture medium again and repeatedly pipette the cells to form a cell suspension. Finally, transfer the cells to a new culture dish according to the number of cells to complete the cell passage.

[0103] SH-SY5Y cells were respectively injected at 1×10 5 Cells were seeded in 96-well plates with 6 replicates per group, 80 μL / well. After seeding, the cells were cultured for 24 h. Positive control drugs fluoxetine, luteolin, hypericin, tetrahydrohypericin, and uralione L were dissolved and diluted to 10 μM. Hyperforidiol E was dissolved and diluted to 5 μM and 10 μM, respectively. Cells were treated with 10 μL / well for 1 h. Then, 10 μL of 25 μM corticosterone was added, and the cells were cultured at 37 °C for 48 h. Finally, 10 μL of CCK-8 was added, and the absorbance at 450 nm was measured after 1 h using a microplate reader, and cell viability was calculated.

[0104] Cell viability (%) = [(Control A - Sample A) / (Control A - Blank A)] × 100%.

[0105] (3) Experimental Results

[0106] The experimental results (Table 2) showed that low and high doses (5 μM and 10 μM) of the compound Hyperforidiol E had a significant protective effect against corticosterone-induced SH-SY5Y neuronal damage. The low dose of Hyperforidiol E was significantly superior to the positive control drug Luyoutai (the first internationally recognized natural antidepressant herbal medicine, 10 μM), Uralione L (10 μM), Hypericin (10 μM), and tetrahydrohypericin (10 μM), while the high dose of Hyperforidiol E was superior to the positive control drug Fluoxetine (a first-line chemical drug for the clinical treatment of depression, 10 μM).

[0107] Table 2. Neuroprotective effects of compounds against corticosterone-induced SH-SY5Y cell damage ( n=5)

[0108]

[0109]

[0110] Note: Compared with the blank group: #### P < 0.0001; Compared with the model group: * P < 0.05 ** P < 0.01, *** P < 0.001

[0111] In summary, Hyperforidiol E described in this invention exhibits significant antidepressant activity, which is superior to first-line antidepressants such as fluoxetine and loratadine, and can be used as a prodrug for treating depressive disorders.

[0112] Application Example 1

[0113] This invention discloses a capsule formulation using Hyperforidiol E as the active pharmaceutical ingredient, the components of which are as follows:

[0114]

[0115] The specific preparation process is as follows:

[0116] Mix Hyperforidiol E, starch, and sodium metabisulfite evenly, add anhydrous ethanol to make a soft material, pass it through a 24-mesh sieve to make granules, dry it, add magnesium stearate, mix well, and fill into capsules.

[0117] Application Example 2

[0118] This invention discloses a granule formulation using the compound Hyperforidiol E as a raw material, the components of which are as follows:

[0119] Hyperforidiol E35.0 mg

[0120] 6.0g of starch

[0121] Sodium bisulfite 0.2g

[0122] Magnesium stearate 0.2g

[0123] Anhydrous ethanol appropriate amount

[0124] Make 100 bags.

[0125] The specific preparation process is as follows:

[0126] Hyperforidiol E was mixed with starch and sodium bisulfite, and then anhydrous ethanol was added to make a soft material. The material was passed through a 24-mesh sieve to make granules, dried, and then magnesium stearate was added. The mixture was then mixed and packaged.

[0127] Application Example 3

[0128] This invention discloses an oral liquid using the compound Hyperforidiol E as a raw material, the components of which are as follows:

[0129]

[0130] The specific preparation process is as follows:

[0131] After the above components are mixed, they can be dispensed using conventional oral liquid preparation methods.

[0132] Application Example 4

[0133] This invention discloses an injectable formulation using Hyperforidiol E as the active pharmaceutical ingredient, the components of which are as follows:

[0134] Hyperforidiol E45.0 mg

[0135] Vitamin C 0.2g

[0136] Sodium chloride 6.0g

[0137] Sodium bicarbonate 0.1g

[0138] 1000mL of water for injection

[0139] Make 100 pieces.

[0140] The specific preparation process is as follows:

[0141] After the above components are mixed, 100 vials can be obtained by using conventional injection preparation methods.

[0142] Application Example 5

[0143] This invention discloses a tablet using Hyperforidiol E and fluoxetine as active pharmaceutical ingredients, the components of which are as follows:

[0144] Hyperforidiol E 20.0mg

[0145] Fluoxetine 0.5g

[0146] 18g of hydroxypropyl methylcellulose

[0147] Talc powder 0.4g

[0148] Lactose 0.2g

[0149] Magnesium stearate 0.2g

[0150] Anhydrous ethanol appropriate amount

[0151] Make 100 pieces.

[0152] The specific preparation process is as follows:

[0153] Mix Hyperforidiol E, fluoxetine, hydroxypropyl methylcellulose, talc, lactose, and magnesium stearate evenly, add anhydrous ethanol to make a soft mass, pass it through a 24-mesh sieve to make granules, dry it, add magnesium stearate, mix well, and compress into tablets.

[0154] Application Example 6

[0155] This invention discloses a capsule formulation using Hyperforidiol E and sertraline as active pharmaceutical ingredients, the components of which are as follows:

[0156] Hyperforidiol E 18.0 mg

[0157] Sertraline 2.0g

[0158] 6.0g of starch

[0159] Sodium metabisulfite 0.2g

[0160] Magnesium stearate 0.2g

[0161] Anhydrous ethanol appropriate amount

[0162] Make 100 pills.

[0163] The specific preparation process is as follows:

[0164] Hyperforidiol E, sertraline, starch, and sodium metabisulfite were mixed and then anhydrous ethanol was added to form a soft mass. The mass was passed through a 24-mesh sieve to form granules, dried, and then magnesium stearate was added. The mixture was then filled into capsules.

[0165] Application Example 7

[0166] This invention discloses an injection using Hyperforidiol E and escitalopram as active pharmaceutical ingredients, the components of which are as follows:

[0167] Hyperforidiol E 30.0mg

[0168] Escitalopram 2.0g

[0169] Vitamin C 0.2g

[0170] Sodium chloride 6.0g

[0171] Sodium bicarbonate 0.1g

[0172] 1000mL of water for injection

[0173] Make 100 pieces.

[0174] The specific preparation process is as follows:

[0175] After the above components are mixed, 100 vials can be obtained by using conventional injection preparation methods.

[0176] The above embodiments are merely examples of several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent.

[0177] It should be noted that those skilled in the art can make various modifications and improvements without departing from the inventive concept, and these all fall within the scope of protection of this invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for preparing a phloroglucinol compound, Hyperforidiol E, characterized in that, Includes the following steps: S1. Take the dried aerial parts of St. John's wort, add solvent and reflux extract, combine the extracts and concentrate to obtain an extract; the solvent is an 88-98 V% ethanol aqueous solution; the mass of the solvent added is 8-10 times that of St. John's wort, the number of reflux extractions is 1-3, and each extraction is 1-3 hours. S2. After suspending the extract in 8-15 times its weight of water, extract with petroleum ether to obtain the extract; S3. The extract was subjected to gradient elution using a petroleum ether-ethyl acetate solution via silica gel column chromatography to obtain fraction E. When identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction E was 0.65-0.

68. Based on a total volume of 100, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution used to elute fraction E was 50:

50. S4. Fraction E was subjected to gradient elution using a petroleum ether-ethyl acetate solution via silica gel column chromatography to obtain fraction E2. When identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction E2 was 0.75-0.

78. The volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution used to elute fraction E2 was 80:

20. S5. Fraction E2 was eluted by gradient elution using a methanol-water solution via MCI column chromatography to obtain fraction E2B. When identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction E2B was 0.47-0.

49. The volume ratio of methanol to water in the methanol-water solution used to elute fraction E2B was 70:

30. S6. The resorcinol compound having the structure shown in formula (1) was separated from fraction E2B by HPLC. The mobile phase used in the HPLC method was acetonitrile-water solution. Fraction E2B was further separated based on the characteristic ultraviolet absorption of resorcinol observed by HPLC analysis. In step S6, the volume ratio of acetonitrile to water in the acetonitrile-water solution was (55:45)-(65:35). （1）。

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