Antidepressant benzenotriol compounds, methods of making and using the same

Abstract

The application belongs to the technical field of medicines, and specifically discloses an anti-depression m-hydroxybenzoic acid compound and a preparation method and application thereof; the m-hydroxybenzoic acid compound Hyperioxides A-F provided by the application has not been reported; the six compounds have good anti-depression effects, and further research shows that the compounds Hyperioxide A, Hyperioxide D, Hyperioxide E and Hyperioxide F have significant protective effects on SH-SY5Y cells induced by corticosterone, and are 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 class of phloroglucinol compounds, their preparation methods, pharmaceutical compositions, and their application in the treatment of antidepressants. Background Technology

[0002] Depression is a common mental health disorder that severely impacts patients' quality of life. Medication is an important treatment for depression; however, traditional medications are prone to tolerance or side effects. Therefore, finding new antidepressants is crucial for improving patients' symptoms and quality of life.

[0003] St. John's wort is a perennial herb belonging to the genus Hypericum in the family Clusiaceae, and its resources are widely distributed. In the search for new drugs to treat depression, the inventors isolated a class of resorcinol compounds, Hyperioxide AF, from the dried aerial parts of St. John's wort and found that Hyperioxide A, Hyperioxide D, Hyperioxide E, and Hyperioxide F have significant antidepressant activity. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a class of phloroglucinol compounds with (2-hydroxy-2-propyl)dihydrofuran substituted at positions 4 and 5, as well as 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 class of antidepressant phloroglucinol compounds having the structures shown in formulas (1), (2), (3), (4), (5), and / or (6):

[0007] (1)

[0009] (2)

[0011] (3)

[0013] (4)

[0015] (5)

[0017]

[0018] (6).

[0019] Tautomers of the aforementioned phloroglucinol compounds or pharmaceutically acceptable salts are also within the scope of protection of this invention.

[0020] In another aspect, the present invention also provides a method for preparing the above-mentioned phloroglucinol compounds, wherein the phloroglucinol compounds are extracted and separated from St. John's wort.

[0021] Furthermore, the method for preparing the resorcinol compound includes the following steps:

[0022] 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.

[0023] S2. After suspending the extract in water, extract it with petroleum ether to obtain the extract;

[0024] S3. The extract was subjected to gradient elution using a petroleum ether-ethyl acetate solution via silica gel column chromatography to obtain fractions C and E. When identified by silica gel thin-layer chromatography, the Rf value for fraction C was 0.75-0.77, and the Rf value for fraction E was 0.65-0.68.

[0025] S4. Fraction E was eluted using a gradient elution with petroleum ether-ethyl acetate solution via silica gel column chromatography to obtain fraction E7. When identified by silica gel thin-layer chromatography, the Rf value for fraction E7 was 0.35-0.44. Fraction C was eluted using a gradient elution with petroleum ether-ethyl acetate solution via silica gel column chromatography to obtain fractions C4 and C9. When identified by silica gel thin-layer chromatography, the Rf value for fraction C4 was 0.72-0.74; the Rf value for fraction C9 was 0.50-0.52.

[0026] S5. Fractions E7, C9, and C4 were eluted using a methanol-water solution via MCI column chromatography to obtain fractions E7A, C9D, and C4E, respectively. When identified by silica gel thin-layer chromatography, the Rf values ​​for fraction E7A were 0.31–0.34; for fraction C9D, 0.58–0.62; and for fraction C4E, 0.70–0.73.

[0027] S6. The resorcinol compounds having structures as shown in formula (1) and / or formula (2) were separated from fraction E7A by HPLC.

[0028] The mobile phase used in the HPLC method is acetonitrile-water solution.

[0029] This application uses acetonitrile-water as the mobile phase and employs HPLC method, utilizing C 18 The chromatographic column was used to prepare phloroglucinol compounds with the structural formulas shown in formulas (1) and (2) in fraction E7A.

[0030] S7. The resorcinol compounds having the structure shown in formula (3) were separated from fraction C9D by HPLC.

[0031] The mobile phase used in the HPLC method is methanol-water solution.

[0032] This application uses methanol-water as the mobile phase and employs HPLC method, utilizing C 18 The chromatographic column was used to prepare phloroglucinol compounds with the structural formula shown in formula (3) in fraction C9D.

[0033] S8. The resorcinol compounds having structures as shown in formula (4), formula (5) and / or formula (6) were separated from fraction C4E by HPLC.

[0034] The mobile phase used in the HPLC method is acetonitrile-water solution.

[0035] This application uses acetonitrile-water as the mobile phase and employs HPLC method, utilizing C 18 The chromatographic column was used to prepare phloroglucinol compounds with the structural formulas shown in formulas (4), (5), and (6) in fraction C4E.

[0036] Further, in step S6, the volume ratio of acetonitrile to water in the acetonitrile-water solution is (55:45) to (65:35). Preferably, the volume ratio of acetonitrile to water in the acetonitrile-water solution is 60:40.

[0037] Further, in step S6, the characteristic ultraviolet absorption (λ) of phloroglucinol observed by HPLC analysis is further analyzed. max =220, 280 nm) Further separation of fraction E7A was performed, including the retention time of phloroglucinol compounds with structural formula as shown in formula (1) for 33-35 min; and the retention time of phloroglucinol compounds with structural formula as shown in formula (2) for 38-40 min.

[0038] Further, in step S7, the volume ratio of methanol to water in the methanol-water solution is (75:25) to (85:15). Preferably, the volume ratio of methanol to water in the methanol-water solution is 80:20.

[0039] Further, in step S7, the characteristic ultraviolet absorption (λ) of phloroglucinol observed by HPLC analysis is... max The fraction C9D was further separated at 220 and 280 nm to obtain phloroglucinol compounds with the structural formula shown in formula (3) with a retention time of 45-55 min.

[0040] Further, in step S8, the volume ratio of acetonitrile to water in the acetonitrile-water solution is (68:32) to (75:25). Preferably, the volume ratio of acetonitrile to water in the acetonitrile-water solution is 72:28.

[0041] Further, in step S8, the characteristic ultraviolet absorption (λ) of phloroglucinol observed by HPLC analysis is... max =220, 280 nm) Further separation of fraction C4E was performed, including the retention time of phloroglucinol compounds with structural formula as shown in formula (4) for 40-45 min; the retention time of phloroglucinol compounds with structural formula as shown in formula (5) for 25-30 min; and the retention time of phloroglucinol compounds with structural formula as shown in formula (6) for 32-38 min.

[0042] Further, in step S2, the extract is suspended in 8-15 times its weight of water and then extracted with petroleum ether.

[0043] In this application, the Rf value is the Rf value of the fluorescent spot observed at scanning wavelengths of 254 nm and 365 nm.

[0044] 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 C (i.e., the fraction corresponding to C) is (75:25) - (85:15).

[0045] 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 50:50.

[0046] Furthermore, 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 C is 80:20.

[0047] The volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution during the washing out of fraction E is 50:50.

[0048] Further, in step S4, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution when washing out fraction E7 (i.e., the fraction corresponding to E7) is (87:13)-(92:8); preferably 10:90.

[0049] Further, in step S4, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution when washing out fraction C9 (i.e., fraction C9 should be) is (15:85)-(25:75); preferably 20:80.

[0050] Further, in step S4, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution when washing out fraction C4 (i.e., the fraction corresponding to C4) is (65:35)-(75:25); preferably 70:30.

[0051] Further, in step S5, the volume ratio of methanol to water in the methanol-water solution when washing out fraction E7A (i.e., the fraction corresponding to E7A) is (65:35)-(72:28); preferably 70:30.

[0052] Further, in step S5, the volume ratio of methanol to water in the methanol-water solution when washing out fraction C9D (i.e., the methanol-water solution corresponding to fraction C9D) is (72:28)-(78:22); preferably 75:25.

[0053] Further, in step S5, the volume ratio of methanol to water in the methanol-water solution when washing out fraction C4E (i.e., the methanol-water solution corresponding to fraction C4E) is (87:13)-(92:8); preferably 90:10.

[0054] Further, in step S1, the solvent is an 88-98 V% 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.

[0055] In another aspect, the present invention provides a pharmaceutical composition containing the above-mentioned phloroglucinol compounds.

[0056] Furthermore, the pharmaceutical composition includes an synergist and a pharmacodynamically acceptable carrier or excipient.

[0057] 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.

[0058] Furthermore, the synergist is one or more of the following substances:

[0059] 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.

[0060] Furthermore, the dosage form of the pharmaceutical composition is tablets, capsules, granules, oral liquids, powders, drops, or micro-pellets.

[0061] In another aspect, the present invention provides the use of the above-mentioned phloroglucinol compounds or the above-mentioned compositions in antidepressant drugs.

[0062] For ease of description, this application defines compounds having formulas (1) to (6) as compounds HyperioxidesA-F.

[0063] Compared with the prior art, the present invention has the following advantages:

[0064] (1) This invention provides hyperioxides AF, a resorcinol compound that has not been previously reported. Experimental results show that the six compounds have good antidepressant effects. In particular, the resorcinol compounds Hyperioxide A, Hyperioxide D, Hyperioxide E, and Hyperioxide F have significant protective effects against corticosterone-induced SH-SY5Y cells, which are significantly better than the positive control drugs fluoxetine and loratadine. They can be used to develop drugs for the treatment of depression.

[0065] (2) The preparation method of phloroglucinol compounds is simple, reproducible and has high extraction purity. Attached Figure Description

[0066] Figure 1 The Hyperioxide A prepared in Example 1 of this invention 1 1H NMR spectrum (400 MHz, CDCl3);

[0067] Figure 2 The Hyperioxide A prepared in Example 1 of this invention 13 C NMR spectrum (100 MHz, CDCl3);

[0068] Figure 3 The HMBC spectrum of Hyperioxide A prepared in Example 1 of this invention;

[0069] Figure 4 The Hyperioxide B prepared in Example 1 of this invention 1 1H NMR spectrum (400 MHz, CDCl3);

[0070] Figure 5 The Hyperioxide B prepared in Example 1 of this invention 13 C NMR spectrum (100 MHz, CDCl3);

[0071] Figure 6 The HMBC spectrum of Hyperioxide B prepared in Example 1 of this invention;

[0072] Figure 7 The Hyperioxide C prepared in Example 2 of this invention 1 1H NMR spectrum (400 MHz, CDCl3);

[0073] Figure 8 The Hyperioxide C prepared in Example 2 of this invention 13 C NMR spectrum (100 MHz, CDCl3);

[0074] Figure 9 The HMBC spectrum of Hyperioxide C obtained in Example 2 of this invention;

[0075] Figure 10 The Hyperioxide D prepared in Example 3 of this invention 1 1H NMR spectrum (400 MHz, CDCl3);

[0076] Figure 11 The Hyperioxide D prepared in Example 3 of this invention 13 C NMR spectrum (100 MHz, CDCl3);

[0077] Figure 12 The HMBC spectrum of Hyperioxide D prepared in Example 3 of this invention;

[0078] Figure 13 The Hyperioxide E prepared in Example 3 of this invention 1 1H NMR spectrum (400 MHz, CDCl3);

[0079] Figure 14 The Hyperioxide E prepared in Example 3 of this invention 13 C NMR spectrum (100 MHz, CDCl3);

[0080] Figure 15 The HMBC spectrum of Hyperioxide E prepared in Example 3 of this invention;

[0081] Figure 16 The Hyperioxide F prepared in Example 3 of this invention 1 1H NMR spectrum (400 MHz, CDCl3);

[0082] Figure 17 The Hyperioxide F prepared in Example 3 of this invention 13 C NMR spectrum (100 MHz, CDCl3);

[0083] Figure 18 The image shows the HMBC spectrum of Hyperioxide F prepared in Example 3 of this invention. Detailed Implementation

[0084] 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.

[0085] 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.

[0086] In the embodiments, unless otherwise specified, all methods used are conventional methods in the art.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] Example 1

[0092] This embodiment discloses a method for preparing phloroglucinol compounds, including the following steps:

[0093] 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 V% aqueous ethanol 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.

[0094] 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).

[0095] S2. After suspending the extract in water, extract with petroleum ether to obtain the extract; preferably, after suspending the extract in 8-15 times its weight of water, extract with petroleum ether to obtain the extract.

[0096] 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).

[0097] 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 at the time of elution of fraction E (i.e., the volume corresponding to fraction E) was (45:55)-(55:45), preferably 50:50.

[0098] 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.

[0099] In this application, the Rf value is the Rf value of the fluorescent spot observed at scanning wavelengths of 254 nm and 365 nm.

[0100] 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 at 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, 11-17, 18-30, 31-35, 36-42, 43-47, 48-50, 51-53, and 54-56. These fractions were identified using silica gel thin-layer chromatography, based on the values ​​at 254 nm and 365 nm. Fluorescent spots with Rf values ​​of 0.81-0.83 (fractions 1-5), 0.78-0.80 (fractions 6-10), 0.75-0.77 (fractions 11-17), 0.72-0.74 (fractions 18-30), 0.65-0.68 (fractions 31-35), 0.64-0.63 (fractions 36-42), 0.57-0.62 (fractions 43-47), 0.52-0.54 (fractions 48-50), 0.46-0.47 (fractions 51-53), and 0.35-0.39 (fractions 54-56) observed at nm were combined with similar fractions 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 31-35.

[0101] The characteristic ultraviolet absorption (λ) of phloroglucinol observed by HPLC analysis max = 220, 280 nm) Select fraction E for the next separation step.

[0102] S4. Fraction E is eluted by gradient elution using a petroleum ether-ethyl acetate solution via silica gel column chromatography to obtain fraction E7. When identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction E7 is 0.35-0.44. The volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution at the time of elution of fraction E7 (i.e., the volume ratio corresponding to fraction E7) is (5:95)-(15:85); preferably 10:90.

[0103] 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: 1-23, 24-35, 36-55, 56-75, 76-85, 86-95, 96-100, and 101-115. These fractions are then identified using silica gel thin-layer chromatography, based on wavelengths of 254 nm and 365 nm. The Rf values ​​of the fluorescent spots observed at nm were 0.79-0.85 (fractions 1-23), 0.75-0.78 (fractions 24-35), 0.71-0.74 (fractions 36-55), 0.63-0.69 (fractions 56-75), 0.50-0.62 (fractions 76-85), 0.45-0.49 (fractions 86-95), 0.35-0.44 (fractions 96-100), and 0.30-0.34 (fractions 101-115). These were combined to obtain fractions E1 to E8; among them, fractions 96-100 were combined to form fraction E7.

[0104] S5. Fraction E7 is eluted by gradient elution using a methanol-water solution via MCI column chromatography to obtain fraction E7A. When identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction E7A is 0.31-0.34. The volume ratio of methanol to water in the methanol-water solution eluted from fraction E7A (i.e., the volume ratio corresponding to fraction E7A) is (65:35)-(72:28); preferably 70:30.

[0105] Specific procedures include: analyzing the characteristic ultraviolet absorption (λ) of phloroglucinol observed by HPLC. max = 220, 280 nm) Selected E7 for further separation. Fraction E7 was subjected to MCI column chromatography, using methanol-water gradient elution with volume ratios of 70:30, 75:25, 80:20, 90:10, and 100:0, and 35 fractions (1-8, 9-15, 16-20, 21-25, and 26-35) 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 254 nm and 365 nm, which were 0.31-0.34 (fractions 1-8), 0.47-0.49 (fractions 9-15), 0.55-0.62 (fractions 16-20), 0.64-0.69 (fractions 21-25), and 0.72-0.75 (fractions 26-35), respectively, five fractions were combined to obtain five fractions, E7A to E7E. Among them, fractions 1-8 are combined to form fraction E7A.

[0106] S6. The resorcinol compounds having structures as shown in formula (1) and / or (2) were separated from fraction E7A by HPLC.

[0107] The mobile phase used in the HPLC method is acetonitrile-water solution.

[0108] This application uses acetonitrile-water as the mobile phase and employs HPLC method, utilizing C 18 The chromatographic column was used to prepare phloroglucinol compounds with the structural formulas shown in formulas (1) and (2) in fraction E7A.

[0109] Wherein, the volume ratio of acetonitrile to water in the acetonitrile-water solution is (55:45) to (65:35). Preferably, the volume ratio of acetonitrile to water in the acetonitrile-water solution is 60:40.

[0110] The characteristic ultraviolet absorption (λ) of phloroglucinol observed by HPLC analysis max = 220, 280 nm) Further separation of fraction E7A was performed, including the retention time of phloroglucinol compounds with the structural formula as shown in formula (1) for 33-35 min (purity: 98%); and the retention time of phloroglucinol compounds with the structural formula as shown in formula (2) for 38-40 min (purity: 99%).

[0111] The physical properties and detection data of compound 1 obtained in Example 1 are as follows:

[0112] White oily substance; its molecular weight is estimated to be 586 based on high-resolution mass spectrometry, and its molecular formula is determined to be C. 35 H 55 O7 has an unsaturation degree of 9.

[0113] like Figure 1 shown 1 In the 1H NMR (400 MHz, CDCl3) spectrum d H The 5.07 (2H, m) signal indicates the presence of two double-bond protons connected to the methylene group in the structure. Furthermore, d H 4.57 (1H, dd, J = 10.8, 5.5 Hz), 3.29 (1H, d, J = 11.1 Hz) indicates two hydroxymethyl signals; 11 groups of methyl hydrogen signals [ d H 1.69(3H, s), 1.64(3H, s), 1.64(3H, s), 1.59(3H, s), 1.38(3H, s), 1.21(3H, s), 1.18(3H, s), 1.14(3H, s), 1.09(3H, d, J = 6.5 Hz), 1.01(3H, d, J= 7.3 Hz), 1.02(3H, s)].

[0114] like Figure 2 shown 13 The C10 NMR (100 MHz, CDCl3) spectrum showed a total of 35 carbon signals. Based on the carbon spectrum information, we inferred that the compound is a phloroglucinol, including 3 carbonyl carbon signals. d C 209.7, 204.6, 192.7), 1 hydroxyl group carbon signal d C 172.9, 5 olefin carbon signals ( d C 132.7, 131.2, 125.0, 121.3, 116.9), 1 quarterly carbon signal d C 48.4, 6 methylene carbon signals ( d C 37.7, 36.0, 30.3, 30.2, 25.4, 22.2), 2 methylene carbon signals ( d C 38.4, 42.2), 10 methyl carbon signals ( d C 27.1, 26.7, 25.8, 25.8, 24.3, 23.7, 21.5, 20.5, 18.0, 17.8, 13.7). In addition, it includes two oxygen-linked tertiary carbon signals (…). d C 90.2, 75.0); 2 oxygen-linked quaternary carbon signals ( d C 73.3, 70.9); quaternary carbon signals of two carbonyl groups ( d C 83.5, 59.4).

[0115] 1 H NMR, 13 The signal assignments for C NMR are shown in Table 1.

[0116] Table 1. NMR data of compound 1 d (ppm)

[0117]

[0118] like Figure 3 In the HMBC spectrum shown, H-27 ( d H 4.57) and C-28 ( d C 70.9), C-29 ( dC 24.3), C-30 ( d C 27.1) and C-4 ( d C Related to 172.9), it was proven that the compound contains a (2-hydroxy-2-propyl)tetrahydrofuran substituent group; H-11 ( d H 2.03) and C-13 ( d C 21.5), C-10 ( d C Related to 209.7), H-17 ( d H 5.07) and C-15 ( d C 36.0), C-16 ( d C 25.4), C-20 ( d C 25.8) related and H-14 ( d H 1.02) and C-15 ( d C 36.0), C-7 ( d C 38.4) Related information indicates that isobutyryl and 4-methyl-3-enpentyl are attached at the C-1 and C-8 positions, respectively; furthermore, H-22 ( d H 3.29) and C-21 ( d C 30.3), C-7 ( d C 38.4), C-23 ( d C 73.3), C-24 ( d C 23.7), C-25 ( d C The long-range correlation between 26.7) proves that 2,3-dihydroxy-3-methylbutyl is linked to the carbon at position 7.

[0119] In summary, the structure of the new compound 1 (compound Hyperioxide A) was determined as follows:

[0120]

[0121] The physical properties and detection data of compound 2 obtained in Example 1 are as follows:

[0122] A white, 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.

[0123] like Figure 4 shown 1 In the H NMR (CDCl3, 400 MHz) spectrum d H The signals at 5.08 (1H, m) and 5.01 (1H, m) indicate the presence of two protons in the double bond connected to the methylene group in the structure. Furthermore, d H 4.59 (1H, dd, J = 10.9, 5.5Hz), 3.30 (1H, d, J = 8.6 Hz) indicates two hydroxymethyl signals; 11 groups of methyl hydrogen signals [ d H 1.70(3H, s), 1.65 (3H, s), 1.63 (3H, s), 1.59(3H, s), 1.37 (3H, s), 1.20 (3H, s), 1.19(3H, s), 1.12 (3H, s), 1.09(3H, d, J = 6.48 Hz), 1.00 (3H, d, J = 6.54 Hz), 1.02(3H,s)].

[0124] like Figure 5 shown 13 The C NMR (CDCl3, 100 MHz) spectrum showed a total of 35 carbon signals, including 3 carbonyl carbon signals. d C 209.7, 204.6, 192.9), 1 hydroxyl group carbon signal d C 173.4, 5 olefin carbon signals ( d C 132.6, 131.4, 124.7, 121.4, 116.7), 1 quarterly carbon signal d C 49.1, 6 methylene carbon signals ( d C 39.5, 36.4, 30.2, 30.6, 25.5, 22.3), 2 methylene carbon signals ( d C 43.2, 42.1), 10 methyl carbon signals ( dC 27.1, 26.9, 25.8, 25.8, 24.2, 22.8, 21.5, 20.5, 18.0, 17.9, 13.8). In addition, it includes two oxygen-bound tertiary carbon signals (…). d C 90.4, 80.2); 2 oxygen-linked quaternary carbon signals ( d C 73.6, 70.9); quaternary carbon signals of two carbonyl groups ( d C 83.6, 59.7).

[0125] 1 H NMR, 13 The signal assignments for C NMR are shown in Table 2.

[0126] Table 2 NMR data of compound 2 d (ppm)

[0127]

[0128] like Figure 6 In the HMBC spectrum shown, H-6b can be observed ( d H 2.56) and C-7 ( d C 43.2), C-8 ( d C 49.1), C-5 ( d C 59.7), C-4 ( d C Related to 173.4), it was further determined that the parent core of the compound is a PPAP-type structure, H-26a ( d H 1.80) and C-5 ( d C 59.7), C-4 ( d C 173.4) related, H-27 ( d H 4.59) and C-28 ( d C 70.9), C-26 ( d C 30.2), C-29 ( d C 27.1), C-30 ( d C Related to 24.2), it was proven that the compound contains a (2-hydroxy-2-propyl)tetrahydrofuran substituent at positions 4 and 5; H-11 ( d H 2.00) and C-10 ( d C 209.7), C-12 ( d C 21.5), C-13 ( d C The correlation between 20.5) indicates the presence of an isobutyryl group in the compound; H-16b ( d H 2.18) and C-17 ( d C 124.7), C-15 ( d C 36.4), C-18 ( d C 131.4), H-15b ( d H 2.08) and C-8 ( d C 43.2), C-7 ( d C 49.1), C-1 ( d C The relevant explanation between 83.6) is that 4-methyl-3-enpentyl is attached at the C-8 position; in addition, H-22 ( d H 3.29) and C-21 ( d C 30.6), C-7 ( d C 43.2), C-23 ( d C 73.6), C-24 ( d C 22.8), C-25 ( d C 26.9), H-32 d C 5.08) and C-34 ( d C 17.9), C-35 ( d C 25.8), C-31 ( d C 22.3), C-3 ( d C The long-range correlation between 116.7) proves that 2,3-dihydroxy-3-methylbutyl and 3-methyl-2-enbutyl are connected to carbons at positions 7 and 3, respectively.

[0129] In summary, the structure of the new compound 2 (compound Hyperioxide B) is determined as follows:

[0130]

[0131] Example 2

[0132] This embodiment discloses a method for preparing phloroglucinol compounds, including the following steps:

[0133] 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; wherein the solvent is 88-98 V% ethanol aqueous solution, and its mass 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.

[0134] Preferably, a 95% ethanol aqueous solution, with a mass of 10 times that of St. John's wort, is added as a solvent for reflux extraction three times, each extraction lasting 2 hours. The extracts are then combined and concentrated to obtain an extract.

[0135] S2. The extract is suspended in 8-15 times its weight of water and then extracted with petroleum ether to obtain the extract. More preferably, the extract is suspended in 10 times its weight of water and then extracted with 1.5 times its volume of petroleum ether, and the extraction is repeated 3 times to obtain the petroleum ether extract.

[0136] S3. The extract was subjected to gradient elution using a petroleum ether-ethyl acetate solution via silica gel column chromatography to obtain fraction C. When identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction C was 0.75-0.77. 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 C (i.e., the fraction corresponding to fraction C) was (75:25)-(85:15), preferably 80:20.

[0137] The specific procedure includes: Petroleum ether extract is subjected to silica gel column chromatography, using a petroleum ether-ethyl acetate gradient elution at volume ratios of 100:0, 90:10, 80:20, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, yielding 56 fractions (1-5, 6-10, 11-17, 18-30, 31-35, 36-42, 43-47, 48-50, 51-53, and 54-56). These fractions are then identified using silica gel thin-layer chromatography, based on the values ​​at 254 nm and 365 nm. Fluorescent spots with Rf values ​​of 0.81-0.83 (fractions 1-5), 0.78-0.80 (fractions 6-10), 0.75-0.77 (fractions 11-17), 0.72-0.74 (fractions 18-30), 0.65-0.68 (fractions 31-35), 0.64-0.63 (fractions 36-42), 0.57-0.62 (fractions 43-47), 0.52-0.54 (fractions 48-50), 0.46-0.47 (fractions 51-53), and 0.35-0.39 (fractions 54-56) observed at nm were combined with similar fractions to obtain 10 fractions, which were named A, B, C, D, E, F, G, H, I, and J, respectively; among them, fraction C was obtained by combining fractions 11-17.

[0138] S4. Fraction C is eluted by gradient elution using a petroleum ether-ethyl acetate solution via silica gel column chromatography to obtain fraction C9. When identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction C9 is 0.50-0.52. The volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution eluted from fraction C9 (i.e., the solution corresponding to fraction C9) is (15:85)-(25:75); preferably 20:80.

[0139] The specific steps include: analyzing the characteristic ultraviolet absorption (λ) of phloroglucinol observed by HPLC. maxFraction C was selected for further separation at wavelengths of 220 and 280 nm. Fraction C was subjected to silica gel column chromatography using a petroleum ether-ethyl acetate gradient elution at volume ratios of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, yielding 111 fractions: 1-15, 16-25, 26-35, 36-45, 46-60, 61-65, 66-73, 74-85, 86-95, 96-98, and 99-111. These fractions were then identified by silica gel thin-layer chromatography at wavelengths of 254 nm and 360 nm. The Rf values ​​of the fluorescent spots observed at nm were 0.85-0.82 (fractions 1-15), 0.79-0.81 (fractions 16-25), 0.75-0.78 (fractions 26-35), 0.72-0.74 (fractions 36-45), 0.68-0.70 (fractions 46-60), 0.65-0.67 (fractions 61-65), 0.63-0.64 (fractions 66-73), 0.60-0.62 (fractions 74-85), 0.50-0.52 (fractions 86-95), 0.45-0.49 (fractions 96-98), and 0.38-0.39 (fractions 99-111). These values ​​were combined to obtain 11 fractions, C1 to C11. Among them, fractions 86-95 were combined to obtain fraction C9.

[0140] S5. Fraction C9 is eluted by gradient elution using a methanol-water solution via MCI column chromatography to obtain fraction C9D. When identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction C9D is 0.58-0.62. The volume ratio of methanol to water in the methanol-water solution eluted from fraction C9D (i.e., the volume ratio corresponding to fraction C9D) is (72:28)-(78:22); preferably 75:25.

[0141] The specific steps are as follows:

[0142] The characteristic ultraviolet absorption (λ) of phloroglucinol observed by HPLC analysis max = 220, 280 nm) C9 was selected for further separation.

[0143] Fraction C9 was subjected to MCI column chromatography, using a methanol-water gradient elution with volume ratios of 60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, and 100:0. Thirty-five fractions (1-4, 5-6, 7-11, 12-17, 18-22, 23-27, 28-31, and 32-35) were collected sequentially. These fractions were then identified using silica gel thin-layer chromatography at wavelengths of 254 nm and 365 nm. The Rf values ​​of the fluorescent spots observed at nm were 0.31-0.34 (fractions 1-4), 0.47-0.49 (fractions 5-6), 0.50-0.52 (fractions 7-11), 0.58-0.62 (fractions 12-17), 0.65-0.69 (fractions 18-22), 0.70-0.73 (fractions 23-27), 0.76-0.78 (fractions 28-31), and 0.80-0.82 (fractions 32-35). These values ​​were combined to obtain eight fractions from C9A to C5H. Fractions 12-17 were further combined to obtain fraction C9D.

[0144] S6. High-adamantane phloroglucinol compounds with the structure shown in formula (3) were separated from fraction C9D by HPLC. The mobile phase used in the HPLC method was methanol-water solution. The volume ratio of methanol to water in the methanol-water solution was (75:25)-(85:15).

[0145] The characteristic ultraviolet absorption (λ) of phloroglucinol observed by HPLC analysis max = 220, 280 nm) Select fraction C9D for further separation; The retention time of the obtained alkyl phloroglucinol compounds is 45-55 min;

[0146] More preferably, the volume ratio of methanol to water in the methanol-water solution is 80:20. Compound 3 was prepared from fraction C9D; the retention time was 49 min (t). R = 49 min, purity 99%).

[0147] In this application, the Rf value is the Rf value of the fluorescent spot observed at scanning wavelengths of 254 nm and 365 nm.

[0148] The physical properties and detection data of compound 3 obtained in Example 2 are as follows:

[0149] A colorless, oily substance, readily soluble in chloroform and methanol. HR-ESI-MS m / z 613.3737 ([M-H]) － The calculated value is 613.4104, which determines the molecular formula of the compound to be C. 37 H 58 O7, its degree of unsaturation is calculated to be 10.

[0150] like Figure 7 shown 1 The 1H NMR (CDCl3, 400 MHz) spectrum showed two olefin proton signals. d H 5.06 (1H, t, J = 7.4 Hz, H-32), 4.94 (1H, m, H-22); d H 3.14 (1H, dd, J = 10.2, 7.4 Hz, H-31a), 3.01 (1H, dd, J = 14.6, 7.4 Hz, H-31b), another d H 3.34 (1H, d, J = 14.6 Hz, H-17), 3.41(1H, dd, J = 6.9, 3.3 Hz, -OCH2CH3) indicates a proton signal bonded to oxygen; 2.23 (1H, m, H-21a), 1.76 (1H, m, H-21b), 2.03 (1H, m, H-15a), 1.83 (1H, overlap, H-15b), 1.79 (1H, m, H-16a), 1.28 (1H, m, H-16b) suggest the presence of methylene groups on three side chains; d H 4.55 (1H, dd, J = 10.9,5.8 Hz, H-27), 2.65 (1H, dd, J = 13.2, 10.9 Hz, H-26a), 1.76 (1H, m, H-26b) suggest the presence of a furan ring fragment formed by the cyclization of isopentenyl and enol carbonyl groups; d H 2.03 (1H, m, H-6a), 1.51 (1H, overlap, H-6b), and 1.61 (1H, overlap, H-7) suggest proton signals on the methylene and methine groups on the ring. d H A value of 1.99 (1H, br m, H-11) suggests the presence of a methine group attached to the carbonyl group; additionally, the spectrum shows proton signals for 11 methyl groups. d H1.68 (6H, br s, H3-35, H3-34), 1.65 (3H, s, H3-24), 1.57 (3H, s, H3-25), 1.38 (3H, s, H3-29), 1.21 (3H, s, H3-30), 1.13 (3H, m, H3-20), 1.13 (3H, m, -OCH2CH3), 1.09 (3H, s, H3-19), 1.04 (3H, s, H3-14), 1.07 (3H, J = 6.4 Hz, H3-12), 0.99 (3H, d, J = 6.4 Hz, H3-13). The above information suggests that this compound may be a PPAP-type compound containing a furan ring.

[0151] like Figure 8 shown 13 The C10 NMR (CDCl3, 100 MHz) spectrum showed a total of 37 carbon signals, and combined with the HSQC spectrum, it was determined that the compound contains 3 carbonyl carbon signals. d C 209.8 (C-10), 204.7 (C-9), 193.0 (C-2); 3 pairs of double bond carbon signals d C 173.2 (C-4), 116.9 (C-3), 133.6 (C-23), 122.4 (C-22), 132.7 (C-33), 121.3 (C-32); 2 oxygen-linked quaternary carbon signals and 2 oxygen-linked tertiary carbon signals d C 90.3 (C-27), 77.5 (C-18), 76.9 (C-17), 71.0 (C-28); a quaternary carbon signal with one carbonyl group attached to three carbonyl groups. d C 83.3 (C-1); 1 methyleneoxy carbon signal d C 56.5 (-OCH2CH3); Quaternary carbon signals on both rings d C 59.6 (C-5), 48.4 (C-8); 2 tertiary carbon signals d C 43.7 (C-7), 42.2 (C-11); 6 secondary carbon signals d C38.1 (C-6), 34.1 (C-15), 30.3 (C-26), 28.3 (C-16), 27.0 (C-21), 22.3 (C-31); 11 methyl carbon signals d C 27.1 (C-29), 24.2 (C-30), 26.1 (C-24), 18.1 (C-25), 25.8 (C-34), 18.0 (C-35), 21.5 (C-20), 20.3 (C-19), 21.6 (C-12); 20.5 (C-13), 16.3 (-OCH2CH3), 13.5 (C-14).

[0152] 1 H NMR, 13 The signal assignments for C NMR are shown in Table 3.

[0153] Table 3 NMR data of compound 3 d (ppm)

[0154]

[0155] In such Figure 9 In the HMBC spectrum shown, H-17 ( d H 3.34) and C-19 ( d C 20.3) and C-16 ( d C 34.9), H-20 ( d H 1.13) and C-19 ( d C 20.3), -OCH2CH3( d C 56.5) and C-17 ( d C 76.9) The presence of long-range correlation, combined with the HSQC spectrum, indicates that the side chain at the C-3 position of this compound is terminally attached to an ethoxy group, and the double bond has undergone rearrangement.

[0156] In summary, the structure of the new compound 3 (compound Hyperioxide C) was determined as follows:

[0157]

[0158] Example 3

[0159] This embodiment discloses a method for preparing phloroglucinol compounds, including the following steps:

[0160] 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 V% aqueous ethanol 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.

[0161] 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).

[0162] S2. After suspending the extract in water, extract it with petroleum ether to obtain the extract;

[0163] Further, in step S2, the extract is suspended in 8-15 times its weight of water and then extracted with petroleum ether.

[0164] 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).

[0165] S3. The extract was subjected to gradient elution using a petroleum ether-ethyl acetate solution via silica gel column chromatography to obtain fraction C. When identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction C was 0.75-0.77. Based on a total volume of 100, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution when fraction C was eluted (i.e., the volume corresponding to fraction C) was (75:25)-(85:15), preferably 80:20.

[0166] In this application, the Rf value is the Rf value of the fluorescent spot observed at scanning wavelengths of 254 nm and 365 nm.

[0167] 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 at 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, 11-17, 18-30, 31-35, 36-42, 43-47, 48-50, 51-53, and 54-56. These fractions were identified using silica gel thin-layer chromatography, based on the values ​​at 254 nm and 365 nm. Fluorescent spots with Rf values ​​of 0.81-0.83 (fractions 1-5), 0.78-0.80 (fractions 6-10), 0.75-0.77 (fractions 11-17), 0.72-0.74 (fractions 18-30), 0.65-0.68 (fractions 31-35), 0.64-0.63 (fractions 36-42), 0.57-0.62 (fractions 43-47), 0.52-0.54 (fractions 48-50), 0.46-0.47 (fractions 51-53), and 0.35-0.39 (fractions 54-56) observed at nm were combined with similar fractions to obtain 10 fractions, which were named A, B, C, D, E, F, G, H, I, and J, respectively; among them, fraction C was obtained by combining fractions 11-17.

[0168] The characteristic ultraviolet absorption (λ) of phloroglucinol observed by HPLC analysis max = 220, 280 nm) Select fraction C for the next step of separation.

[0169] S4. Fraction C is eluted by gradient elution using a petroleum ether-ethyl acetate solution via silica gel column chromatography to obtain fraction C4. When identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction C4 is 0.72-0.74. The volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution eluted from fraction C4 (i.e., the fraction corresponding to C4) is (65:35)-(75:25); preferably 70:30.

[0170] The specific steps include: analyzing the characteristic ultraviolet absorption (λ) of phloroglucinol observed by HPLC. maxFraction C was selected for further separation at wavelengths of 220 and 280 nm. Fraction C was subjected to silica gel column chromatography using a petroleum ether-ethyl acetate gradient elution at volume ratios of 100:0, 90:10, 80:20, 70:30, 60:40, 40:60, 50:50, 30:70, 20:80, 10:90, and 0:100, yielding 111 fractions: 1-15, 16-25, 26-35, 36-45, 46-60, 61-65, 66-73, 74-85, 86-95, 96-98, and 99-111. These fractions were then identified by silica gel thin-layer chromatography at wavelengths of 254 nm and 360 nm. The Rf values ​​of the fluorescent spots observed at nm were 0.85-0.82 (fractions 1-15), 0.79-0.81 (fractions 16-25), 0.75-0.78 (fractions 26-35), 0.72-0.74 (fractions 36-45), 0.68-0.70 (fractions 46-60), 0.65-0.67 (fractions 61-65), 0.63-0.64 (fractions 66-73), 0.60-0.62 (fractions 74-85), 0.50-0.52 (fractions 86-95), 0.45-0.49 (fractions 96-98), and 0.38-0.39 (fractions 99-111). These values ​​were combined to obtain 11 fractions, C1 to C11. Among them, fractions 36-45 were combined to obtain fraction C4.

[0171] S5. Fraction C4 is eluted by gradient elution using a methanol-water solution via MCI column chromatography to obtain fraction C4E. When identified by silica gel thin-layer chromatography, the Rf value corresponding to fraction C4E is 0.70-0.73. The volume ratio of methanol to water in the methanol-water solution eluted from fraction C4E (i.e., the volume ratio corresponding to fraction C4E) is (87:13)-(92:8); preferably 90:10.

[0172] The specific operations include:

[0173] The characteristic ultraviolet absorption (λ) of phloroglucinol observed by HPLC analysis max = 220, 280 nm) C4 was selected for further separation.

[0174] Fraction C4 was subjected to MCI column chromatography, using a methanol-water gradient elution with volume ratios of 70:30, 75:25, 80:20, 85:15, 90:10, 95:5, and 100:0. Thirty-five fractions (1-7, 8-12, 13-17, 18-23, 24-26, 27-31, and 32-35) were collected sequentially. These fractions were then identified using silica gel thin-layer chromatography at wavelengths of 254 nm and 365 nm. The Rf values ​​of the fluorescent spots observed at nm were 0.31-0.34 (fractions 1-7), 0.47-0.49 (fractions 8-12), 0.58-0.62 (fractions 13-17), 0.65-0.69 (fractions 18-23), 0.70-0.73 (fractions 24-26), 0.76-0.78 (fractions 27-31), and 0.80-0.82 (fractions 32-35). These were combined to obtain seven fractions, C4A to C4G. Fractions 24-26 were further combined to obtain fraction C4E.

[0175] S6. The resorcinol compounds having structures as shown in formula (4) and / or (5) and / or (6) were isolated from fraction C4E by HPLC.

[0176] The mobile phase used in the HPLC method is acetonitrile-water solution.

[0177] This application uses acetonitrile-water as the mobile phase and employs HPLC method, utilizing C 18 The chromatographic column was used to prepare phloroglucinol compounds with the structural formulas shown in formulas (4), (5), and (6) in fraction C4E.

[0178] Wherein, the volume ratio of acetonitrile to water in the acetonitrile-water solution is (68:32) to (75:25). Preferably, the volume ratio of acetonitrile to water in the acetonitrile-water solution is 72:28.

[0179] The characteristic ultraviolet absorption (λ) of phloroglucinol observed by HPLC analysis max = 220, 280 nm) Further separation of the C4E fraction included phloroglucinol compounds with the structural formula as shown in formula (4) with a retention time of 40-45 min (purity 98%); phloroglucinol compounds with the structural formula as shown in formula (5) with a retention time of 25-30 min (purity 99%); and phloroglucinol compounds with the structural formula as shown in formula (6) with a retention time of 32-38 min (purity 99%).

[0180] The physical properties and detection data of compound 4 obtained in Example 3 are as follows:

[0181] A colorless, oily substance, readily soluble in chloroform and methanol. HR-ESI-MS m / z567.3654 ([M + H]) + The calculated value is 567.3641, which determines the molecular formula of the compound to be C. 35 H 50 O6 has an unsaturation degree of 11.

[0182] like Figure 10 shown 1 H NMR (CDCl3, 400 MHz) spectrum showed the presence of a pair of carbon-coupled olefin proton signals. d H 6.10 (1H, d, J = 4.0 Hz, H-19a), 5.78 (1H, d, J = 4.0 Hz, H-19b); proton signals on the two double bonds d H 5.07 (1H, br s, H-32), 4.92 (1H, br s, H-22); d H 3.13 (1H, dd, J = 13.9,6.0 Hz, H-31a), 3.02 (1H, dd, J = 13.9, 6.0 Hz, H-31b), 2.89 (1H, ddt, J = 16.7,11.7, 4.6, Hz, H-16a), 2.66 (1H, ddt, J = 16.7, 11.7, 4.6, Hz, H-16b), 2.19 (1H, overlap, H-21a), 1.75 (1H, overlap, H-21b) suggest the presence of a methylene group connected to three double bonds; d H 4.57 (1H, overlap, H-27), 2.66 (1H, overlap, H-26a), 1.77 (1H, dd, J = 13.1, 5.3Hz, H-26b) suggests the presence of a furan ring fragment formed by the cyclization of isopentenyl and enol carbonyl groups; d H 2.05 (1H, d, J =13.1 Hz, H-6a), 1.53 (1H, overlap, H-6b), 1.60 (1H, overlap, H-7) suggest proton signals on the methylene and methine groups on the ring; d H2.01 (1H, br m, H-11) indicates the presence of a methine group attached to the carbonyl group; additionally, the spectrum shows proton signals for 10 methyl groups. d H 1.83 (3H, br s, H3-20), 1.69 (6H, br s, H3-24, H3-35), 1.65 (3H, br s, H3-34), 1.57 (3H, s, H3-25), 1.38 (3H, s, H3-29), 1.21 (3H, s, H3-30), 1.07 (6H, overlap, H3-12, H3-14), 1.00 (3H, d, J = 6.1 Hz, H3-13). The above information suggests that the compound may be a phloroglucinol compound containing a furan ring.

[0183] like Figure 11 shown 13 The C10 NMR (CDCl3, 100 MHz) spectrum showed a total of 35 carbon signals. Based on the proton NMR data, it is inferred that this compound contains 4 carbonyl carbon signals. d C 210.1 (C-10), 204.3 (C-9), 203.0 (C-17), 193.1 (C-2); 4 pairs of double bond carbon signals d C 173.3 (C-4), 116.9 (C-3), 144.2 (C-18), 125.4 (C-19), 133.9 (C-23), 122.3 (C-22), 132.9 (C-33), 121.2 (C-32); one oxygen-linked quaternary carbon signal and one oxygen-linked tertiary carbon signal. d C 90.4 (C-27), 70.9 (C-28); a quaternary carbon signal attached to three carbonyl groups. d C 82.8 (C-1); quaternary carbon signals on two rings d C 59.6 (C-5), 48.5 (C-8); 2 tertiary carbon signals d C 42.6 (C-7), 42.2 (C-11); 6 secondary carbon signals d C 38.2 (C-6), 34.9 (C-16), 31.9 (C-15), 30.3 (C-26), 27.0 (C-21), 22.3 (C-31); 10 methyl carbon signals dC 27.1 (C-29), 24.2 (C-30), 26.1 (C-24), 18.2 (C-25), 25.8 (C-34), 17.8(C-35), 21.5 (C-12), 20.6 (C-13), 18.0 (C-20), 14.4 (C-14).

[0184] 1 H NMR, 13 The signal assignments for C NMR are shown in Table 4 below.

[0185] Table 4 NMR data of compound 4 d (ppm)

[0186]

[0187] like Figure 12 As shown in the HMBC spectrum, H-14 ( d H 1.06) and C-4 ( d C 42.6), C-15 ( d C 34.9) and C-16 ( d C 31.9) Remotely related, and H-20 ( d H 1.82) is also related to C-17 ( d C 203.0), C-18 d C 125.4) and C-19 ( d C 144.3) has a long-range correlation, indicating that the only difference between this compound and furohyperforin is that the isopentenyl group attached to the C-3 position of this compound is further oxidized to form a carbonyl double bond conjugated structure.

[0188] In summary, the structure of the new compound 4 (Hyperioxide D) was determined as follows:

[0189]

[0190] The physical properties and detection data of compound 5 obtained in Example 3 are as follows:

[0191] Colorless oily substance; HR-ESI-MS m / z 629.3687 ([M+HCOO]) －The calculated value is 629.3690, which determines the molecular formula of the compound to be C. 35 H 52 O7, its degree of unsaturation is calculated to be 10.

[0192] like Figure 13 shown 1 The 1H NMR (CDCl3, 400 MHz) spectrum showed signals from four olefin protons. d H 5.74 (1H,dt, J = 15.2, 7.6 Hz, H-16), 5.50 (1H, d, J = 15.2 Hz, H-17), 5.05 (1H, br t, J =7.4, Hz, H-32), 4.93 (1H, br d, J = 7.0 Hz, H-22); and δH 3.11 (1H, dd, J = 14.4, 7.2Hz, H-31a), 3.02 (1H, dd, J = 14.4, 7.2 Hz, H-31b), 1.96 (1H, m, H-15a), 1.69 (1H, overlap, H-15b), 2.22 (1H, overlap, H-21a), 1.76 (1H, m, H-21b) suggest the presence of 3 methylene groups connected to the double bond. d H 4.56 (1H, dd, J = 10.0, 5.8 Hz, H-27), 2.64 (1H, overlap, H-26a), 1.78 (1H, dd, J = 13.2, 5.8 Hz, H-26b) suggests the presence of a furan ring fragment formed by the cyclization of isopentenyl and enol carbonyl groups; d H 2.06 (1H, dd, J = 13.6, 3.6 Hz, H-6a), 1.53 (1H, overlap, H-6b), 1.60 (1H, m, H-7) suggest proton signals on the methylene and methine groups on the ring; d H 2.13 (1H, br m, H-11) suggests the presence of a methine group attached to the carbonyl group; additionally, the spectrum shows proton signals for 11 methyl groups. d H1.29 (6H, br s, H3-19, H3-20), 1.69 (6H, br s, H3-24, H3-35), 1.64 (3H, br s, H3-34), 1.56 (3H, s, H3-25), 1.38 (3H, s, H3-29), 1.21 (3H, s, H3-30), 1.06 (3H, overlap, H3-12, H3-14), 0.99 (3H, d, J = 6.0 Hz, H3-13). The above information suggests that the compound may be a PPAP-type compound containing a furan ring.

[0193] like Figure 14 shown 13 The C10 NMR (CDCl3, 100 MHz) spectrum showed a total of 35 carbon signals. Combined with the proton NMR data, it was determined that the compound contains 3 carbonyl carbon signals. d C 209.7 (C-10), 204.4 (C-9), 192.9 (C-2); 4 pairs of double bond carbon signals d C 173.3 (C-4), 116.9 (C-3), 135.7 (C-17), 129.3 (C-16), 134.1 (C-23), 122.1 (C-22), 132.9 (C-33), 121.1 (C-32); one oxygen-linked quaternary carbon signal and one oxygen-linked tertiary carbon signal. d C 90.4 (C-27), 71.0 (C-28); a quaternary carbon signal attached to three carbonyl groups. d C 82.5 (C-1); a carbon signal linked to a peroxy group. d C 81.9 (C-18); quaternary carbon signals on two rings d C 59.7 (C-5), 48.7 (C-8); 2 tertiary carbon signals d C 41.7 (C-7), 42.3 (C-11); 5 secondary carbon signals d C 38.3 (C-6), 39.6 (C-15), 30.3 (C-26), 27.0 (C-21), 22.2 (C-31); 11 methyl carbon signals d C27.1 (C-29), 24.8 (C-30), 26.1 (C-24), 18.2 (C-25), 25.8 (C-34), 24.2 (C-20), 24.1 (C-19), 18.0 (C-35), 21.5 (C-12), 20.6 (C-13), 15.3 (C-14).

[0194] 1 H NMR, 13 The signal assignments for C NMR are shown in Table 5.

[0195] Table 5 NMR data of compound 5 d (ppm)

[0196]

[0197] like Figure 15 In the HMBC spectrum shown, H-19 ( d H 1.29) and C-16 ( d C 129.3) and C-18 ( d C 81.9) The presence of a long-range correlation indicates that the peroxy group in this compound is linked at the end of the side chain attached to the C-3 position.

[0198] In summary, the structure of the new compound 5 (compound Hyperioxide E) was determined as follows:

[0199]

[0200] The physical properties and detection data of compound 6 obtained in Example 3 are as follows:

[0201] Colorless oily substance; HR-ESI-MS m / z 613.3735 ([M+HCOO]) － The calculated value is 613.3740, which determines the molecular formula of the compound to be C. 35 H 52 O6 has an unsaturation degree of 10.

[0202] like Figure 16 shown 1 The 1H NMR (CDCl3, 400 MHz) spectrum showed signals from four olefin protons. d H 5.71 (1H,dt, J = 15.6, 7.4 Hz, H-16), 5.59 (1H, d, J= 15.6 Hz, H-17), 5.07 (1H, br t, J =7.2, Hz,H-32), 4.89 (1H, br d, J = 7.2 Hz, H-22); and δH 3.11 (1H, dd, J = 14.4, 7.4Hz, H-31a), 3.02 (1H, dd, J = 14.4, 7.4 Hz, H-31b), 2.00 (1H, m, H-15a), 1.68 (1H, overlap, H-15b), 2.25 (1H, overlap, H-21a), 1.78 (1H, m, H-21b) suggest the presence of 3 methylene groups connected to the double bond; d H 4.55 (1H, dd, J = 10.8, 5.6 Hz, H-27), 2.65 (1H, dd, J = 13.0,10.8 Hz, H-26a), 1.77 (1H, dd, J = 13.0, 5.6 Hz, H-26b) suggests the presence of a furan ring fragment formed by the cyclization of isopentenyl and enol carbonyl groups; d H 2.03 (1H, dd, J = 13.6, 3.6 Hz, H-6a), 1.55 (1H, overlap, H-6b), 1.59 (1H, m, H-7) suggest proton signals on the methylene and methine groups on the ring; d H 2.11 (1H, br m, H-11) suggests the presence of a methine group attached to the carbonyl group; additionally, the spectrum shows proton signals for 11 methyl groups. d H 1.27 (6H, br s, H3-19, H3-20), 1.70 (3H, br s, H3-24), 1.68 (3H, br s, H3-35), 1.65 (3H, br s, H3-34), 1.55 (3H, s, H3-25), 1.38 (3H, s, H3-29), 1.21 (3H,s, H3-30), 1.05 (3H,s, H3-14), 1.09 (3H, J = 6.5 Hz, H3-12), 0.99 (3H, d, J= 6.5Hz, H3-13). The above information suggests that the compound may be a PPAP-type compound containing a furan ring.

[0203] like Figure 17 shown 13 The C10 NMR (CDCl3, 100 MHz) spectrum showed a total of 35 carbon signals. Combined with the proton NMR data, it was determined that the compound contains 4 carbonyl carbon signals. d C 210.0 (C-10), 204.2 (C-9), 193.1 (C-2); 4 pairs of double bond carbon signals d C 173.6 (C-4), 116.8 (C-3), 140.2 (C-17), 125.1 (C-16), 133.7 (C-23), 122.4 (C-22), 132.8 (C-33), 121.2 (C-32); one oxygen-linked quaternary carbon signal and two oxygen-linked tertiary carbon signals. d C 90.3 (C-27), 71.0 (C-28), 70.9 (C-18); a quaternary carbon signal with one carbonyl group attached to three carbonyl groups. d C 82.9 (C-1); quaternary carbon signal on two rings d C 59.6 (C-5), 48.8 (C-8); 2 tertiary carbon signals d C 42.2 (C-7), 42.8 (C-11); 5 secondary carbon signals d C 38.4 (C-6), 39.6 (C-15), 30.3 (C-26), 27.2 (C-21), 22.3 (C-31); 11 methyl carbon signals d C 27.0 (C-29), 24.2 (C-30), 26.0 (C-24), 18.2 (C-25), 25.8 (C-34), 29.7 (C-20), 29.8(C-19), 18.0 (C-35), 21.6 (C-12), 20.6 (C-13), 14.6 (C-14).

[0204] 1 H NMR, 13 The signal assignments for C NMR are shown in Table 6.

[0205] Table 6 NMR data of compound 6 d (ppm)

[0206]

[0207] like Figure 18 In the HMBC spectrum shown, H-19 ( d H 1.27) and C-17 ( d C 140.4), C-18 ( d C 70.9) and C-20 ( d C 29.7) The presence of long-range correlation indicates that the side chain at the C-3 position of this compound is terminally attached to a hydroxyl group, and that the double bond has rearranged.

[0208] In summary, the structure of the new compound 6 (compound Hyperioxide F) was determined as follows:

[0209]

[0210] Comparative Example 1

[0211] Comparative Example 1 of this invention provides a method for preparing phloroglucinol compounds, the steps of which are similar to those in Example 1, except that in step S5, when fraction E7 is subjected to MCI column chromatography, a methanol-water solution with a methanol-to-water volume ratio of less than 65:35 or greater than 72:28 is used for elution. The results show that the purity of compound 1 is not higher than 70%, and the purity of compound 2 is not higher than 75%.

[0212] Comparative Example 2

[0213] Comparative Example 2 of this invention provides a method for preparing phloroglucinol compounds, the steps of which are similar to those in Example 2, except that in step S4, when fraction C is subjected to silica gel column chromatography, a petroleum ether-ethyl acetate gradient elution with a volume ratio of less than 15:85 and more than 25:75 is used. The results show that the purity of the obtained compound 3 is not higher than 50%.

[0214] Comparative Example 3

[0215] Comparative Example 3 of this invention provides a method for preparing phloroglucinol compounds, the steps of which are similar to those of Example 3, except that in step S4, when fraction C is subjected to silica gel column chromatography, a petroleum ether-ethyl acetate gradient elution with a volume ratio of less than 65:35 and greater than 75:25 is used. The results show that the purity of compound 4 is not higher than 50%, compound 5 is not higher than 40%, and compound 6 is not higher than 45%.

[0216] 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.

[0217] Test case

[0218] This experimental example discloses the antidepressant activity of the above compounds in corticosterone-induced SH-SY5Y cells (a commonly used cell model for antidepressants) using Hyperioxide A, Hyperioxide B, Hyperioxide C, Hyperioxide D, Hyperioxide E, and Hyperioxide F.

[0219] (1) Experimental materials and instruments

[0220] Human neuroblastoma SH-SY5Y was purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences.

[0221] DMEM medium, PBS buffer, fetal bovine serum (FBS) and trypsin were all purchased from Gibco, USA.

[0222] Fluoxetine (Shanghai Yuanye Biotechnology Co., Ltd.);

[0223] Luyutai (Dr. Wima Schuppe Pharmaceuticals, Germany).

[0224] CCK-8 Cell Viability Assay Kit (Elabscience);

[0225] 96-well plate (Corning Corporation, USA);

[0226] Carbon dioxide cell incubator (Thermo Fisher Scientific, USA).

[0227] Full-wavelength microplate reader (Thermo Fisher Scientific, USA);

[0228] BIOFUGE STRATOS centrifuge (Thermo Fisher Scientific, USA);

[0229] IX73 inverted electron microscope (Olympus Corporation, Japan);

[0230] Pipettes (Eppendorf, Germany);

[0231] Electronic balance (Sartorius GmbH, Germany);

[0232] Clean benches, centrifuge tubes, straws and other related consumables.

[0233] (2) Establishment and evaluation of a corticosterone-damaged SH-SY5Y cell model

[0234] 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 15 mL 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 10 mL of culture medium, mix well, resuspend the cells, transfer them to a 10 mL culture dish, and incubate in a 37°C, 5% CO2 incubator.

[0235] 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 800 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, according to the number of cells, transfer them to a new culture dish to complete the cell passage.

[0236] 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 inhibitors fluoxetine, luteolin, hypericin, and tetrahydrohypericin were dissolved and diluted to 10 μM. Hyperioxide A, Hyperioxide B, Hyperioxide C, Hyperioxide D, Hyperioxide E, and Hyperioxide F were dissolved and diluted to 5 μM and 10 μM, respectively. Cells were treated with 10 μL / well for 1 h, followed by the addition of 10 μL of 25 μM corticosterone. The cells were cultured at 37°C for 48 h, and then 10 μL of a CCK-8 assay kit was added. After 1 h, the absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated.

[0237] Cell viability (%) = [(A control - A sample) / (A control - A blank)] × 100%.

[0238] (3) Experimental results

[0239] The experimental results (Table 7) showed that all compounds Hyperioxides AF had antidepressant effects. Particularly significant, compared with the model group, low and high doses (5 μM, 10 μM) of compounds Hyperioxide A, Hyperioxide D, and Hyperioxide E exhibited significant protective effects against corticosterone-induced SH-SY5Y neuronal damage. At a concentration of 5 μM, Hyperioxide A and Hyperioxide D were superior to the positive control drug Luyoutai (the first internationally recognized natural antidepressant herbal drug, 10 μM), and significantly superior to Hypericin (10 μM) and tetrahydrohypericin (10 μM). At a concentration of 10 μM, the antidepressant activity of Hyperioxide A was significantly superior to that of the positive control drug Fluoxetine (a first-line chemical drug for the clinical treatment of depression, 10 μM).

[0240] Table 7. Neuroprotective effects of compounds against corticosterone-induced SH-SY5Y cell damage ( ±s, n=5)

[0241]

[0242] Note: Compared with the blank group: #### P <0.0001: Compared with the model group * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001

[0243] In summary, the phloroglucinol compounds Hyperioxides AF described in this invention have good antidepressant effects; particularly significantly, Hyperioxide A, Hyperioxide D, Hyperioxide E, and Hyperioxide F exhibit significant antidepressant activity, which is superior to first-line antidepressant drugs fluoxetine and loratadine, and can be used as drugs for the treatment of depression.

[0244] Application Example 1

[0245] This invention discloses a capsule formulation using Hyperioxide D as the active pharmaceutical ingredient, the components of which are as follows:

[0246] Hyperioxide D 18.0 mg

[0247] Starch 6.0 g

[0248] Sodium metabisulfite 0.2 g

[0249] Magnesium stearate 0.2 g

[0250] Anhydrous ethanol appropriate amount

[0251] Make 100 pills.

[0252] The specific preparation process is as follows:

[0253] Mix Hyperioxide D, 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.

[0254] Application Example 2

[0255] This invention discloses a granule formulation using compound Hyperioxide A as the active pharmaceutical ingredient, the components of which are as follows:

[0256] Hyperioxide A 35.0 mg

[0257] Starch 6.0 g

[0258] Sodium bisulfite 0.2 g

[0259] Magnesium stearate 0.2 g

[0260] Anhydrous ethanol appropriate amount

[0261] Make 100 bags.

[0262] The specific preparation process is as follows:

[0263] Hyperioxide A 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 form granules, dried, and then magnesium stearate was added. The mixture was then mixed and packaged.

[0264] Application Example 3

[0265] This invention discloses an oral liquid using the compound Hyperioxide D as a raw material, the components of which are as follows:

[0266] Hyperioxide D 25.0 mg

[0267] 3.0 g sucrose

[0268] Sodium bisulfite 0.2 g

[0269] Methylparaben 0.2 g

[0270] Sodium bicarbonate 0.1 g

[0271] 1000 mL of water for injection

[0272] Make 100 pieces.

[0273] The specific preparation process is as follows:

[0274] After the above components are mixed, they can be dispensed using conventional oral liquid preparation methods.

[0275] Application Example 4

[0276] This invention discloses an injectable formulation using Hyperioxide D as the active pharmaceutical ingredient, the components of which are as follows:

[0277] Hyperioxide D45.0 mg

[0278] Vitamin C 0.2 g

[0279] Sodium chloride 6.0 g

[0280] Sodium bicarbonate 0.1 g

[0281] 1000mL of water for injection

[0282] Make 100 pieces.

[0283] The specific preparation process is as follows:

[0284] After the above components are mixed, 100 vials can be obtained by using conventional injection preparation methods.

[0285] Application Example 5

[0286] This invention discloses a tablet using compounds Hyperioxide E and fluoxetine as active pharmaceutical ingredients, the composition of which is as follows:

[0287] Hyperioxide E20.0 mg

[0288] Fluoxetine 0.5 g

[0289] Hydroxypropyl methylcellulose 18 g

[0290] 0.4 g talc

[0291] Lactose 0.2 g

[0292] Magnesium stearate 0.2 g

[0293] Anhydrous ethanol appropriate amount

[0294] Make 100 pieces.

[0295] The specific preparation process is as follows:

[0296] Mix Hyperioxide 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.

[0297] Application Example 6

[0298] This invention discloses a capsule formulation using compounds Hyperioxide F and sertraline as active pharmaceutical ingredients, the components of which are as follows:

[0299] Hyperioxide F 18.0 mg

[0300] Sertraline 2.0 g

[0301] Starch 2.0 g

[0302] Sodium metabisulfite 0.2 g

[0303] Magnesium stearate 0.2 g

[0304] Anhydrous ethanol appropriate amount

[0305] Make 100 pills.

[0306] The specific preparation process is as follows:

[0307] Hyperioxide F, 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.

[0308] Application Example 7

[0309] This invention discloses an injection using compounds Hyperioxide A and escitalopram as active pharmaceutical ingredients, the components of which are as follows:

[0310] Hyperioxide A 30.0 mg

[0311] Escitalopram 2.0 g

[0312] Vitamin C 0.2 g

[0313] Sodium chloride 6.0 g

[0314] Sodium bicarbonate 0.1 g

[0315] 1000 mL of water for injection

[0316] Make 100 pieces.

[0317] The specific preparation process is as follows:

[0318] After the above components are mixed, 100 vials can be obtained by using conventional injection preparation methods.

[0319] 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.

[0320] 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. An antidepressant phloroglucinol compound, characterized in that, It has a structure as shown in Equation (1), Equation (4), Equation (5) and / or Equation (6): （1） （4） （5） （6）。 2. A method for preparing the phloroglucinol compound as described in claim 1, characterized in that, The phloroglucinol compounds were extracted and isolated from the aerial parts of St. John's wort. The method for preparing the resorcinol compounds includes the following steps: 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. S2. After suspending the extract in water, extract it 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 fractions C and E. When identified by silica gel thin-layer chromatography, the Rf value for fraction C was 0.75-0.77, and the Rf value for 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 when fraction C is washed out is (75:25) - (85:15). The volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution when fraction E is washed out is (45:55) - (55:45). S4. Fraction E was eluted using a gradient elution with petroleum ether-ethyl acetate solution via silica gel column chromatography to obtain fraction E7. When identified by silica gel thin-layer chromatography, the Rf value for fraction E7 was 0.35-0.

44. Fraction C was eluted using a gradient elution with petroleum ether-ethyl acetate solution via silica gel column chromatography to obtain fractions C4 and C9. When identified by silica gel thin-layer chromatography, the Rf value for fraction C4 was 0.72-0.74; the Rf value for fraction C9 was 0.50-0.

52. The volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution when the eluent E7 is washed out is (5:95)-(15:85). The volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution when the C9 fraction is washed out is (15:85)-(25:75). The volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution when the C4 fraction is washed out is (65:35)-(75:25). S5. Fractions E7, C9, and C4 were eluted using a methanol-water solution via MCI column chromatography to obtain fractions E7A, C9D, and C4E, respectively. When identified by silica gel thin-layer chromatography, the Rf values ​​for fraction E7A were 0.31–0.34; for fraction C9D, 0.58–0.62; and for fraction C4E, 0.70–0.

73. The volume ratio of methanol to water in the methanol-water solution when washing out fraction E7A is (65:35)-(72:28). The volume ratio of methanol to water in the methanol-water solution when washing out fraction C9D is (72:28)-(78:22). The volume ratio of methanol to water in the methanol-water solution when washing out fraction C4E is (87:13)-(92:8). S6. The resorcinol compounds having structures as shown in formula (1) and / or (2) were separated from fraction E7A by HPLC. The mobile phase used in the HPLC method is an acetonitrile-water solution; the volume ratio of acetonitrile to water in the acetonitrile-water solution is (55:45) to (65:35). S7. The resorcinol compounds having the structure shown in formula (3) were separated from fraction C9D by HPLC. The mobile phase used in the HPLC method is a methanol-water solution; the volume ratio of methanol to water in the methanol-water solution is (75:25) to (85:15). S8. The resorcinol compounds having structures as shown in formula (4), formula (5) and / or formula (6) were separated from fraction C4E by HPLC. The mobile phase used in the HPLC method is an acetonitrile-water solution; the volume ratio of acetonitrile to water in the acetonitrile-water solution is (68:32)-(75:25).

3. The preparation method according to claim 2, characterized in that, In step S6, fraction E7A is further separated based on the characteristic ultraviolet absorption of phloroglucinol observed by HPLC analysis, including phloroglucinol compounds with the structural formula shown in formula (1) with a retention time of 33-35 min. The retention time of compounds including phloroglucinols with the structural formula shown in formula (2) is 38-40 min.

4. The preparation method according to claim 2, characterized in that, In step S7, the C9D fraction is further separated based on the characteristic ultraviolet absorption of phloroglucinol observed by HPLC analysis, and the retention time of the phloroglucinol compound with the structural formula shown in formula (3) is 45-55 min.

5. The preparation method according to claim 2, characterized in that, In step S8, the C4E fraction is further separated based on the characteristic ultraviolet absorption of resorcinol observed by HPLC analysis, including the resorcinol compounds with the structural formula shown in formula (4) having a retention time of 40-45 min. The retention time of phloroglucinol compounds, including those with the structural formula shown in formula (5), is 25-30 min; The retention time of phloroglucinol compounds, including those with the structural formula shown in formula (6), is 32-38 min.

6. The preparation method according to claim 2, characterized in that, In step S2, the extract is suspended in 8-15 times its weight of water and then extracted with petroleum ether.

7. The preparation method according to claim 2, characterized in that, 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 C is 80:

20. And / or, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution during the washing out of fraction E is 50:

50.

8. The preparation method according to claim 2, characterized in that, In step S4, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution when washing out fraction E7 is 10:

90. And / or, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution when washing out fraction C9 is 20:80; And / or, the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solution during the washing out of fraction C4 is 70:

30.

9. The preparation method according to claim 2, characterized in that, In step S5, the volume ratio of methanol to water in the methanol-water solution when washing out fraction E7A is 70:

30. And / or, the volume ratio of methanol to water in the methanol-water solution when washing out fraction C9D is 75:25; And / or, the volume ratio of methanol to water in the methanol-water solution when washing out fraction C4E is 90:

10.

10. The preparation method according to claim 2, characterized in that, In step S1, the solvent is an 88-98% V% 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.

11. A pharmaceutical composition, characterized in that, It includes the phloroglucinol compounds as described in claim 1.

12. The pharmaceutical composition according to claim 11, characterized in that, The pharmaceutical composition also includes an synergist and a pharmacodynamically acceptable carrier or excipient.

13. The pharmaceutical composition according to claim 12, characterized in that, The synergist is one or more of the following substances: 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.

14. The pharmaceutical composition according to claim 11, characterized in that, The dosage form of the pharmaceutical composition is tablets, capsules, granules, oral liquids, powders, drops, or micro-pellets.

15. The use of the phloroglucinol compound of claim 1 and / or the pharmaceutical composition of any one of claims 11-14 in the preparation of antidepressant drugs.

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