Preparation method of diterpene alkaloid 3-acetylaconitine

Abstract

The present invention relates to the field of medical technology, and discloses a diterpene alkaloid 3-acetylaconitine, a preparation method, a pharmaceutical composition and an application thereof. 19 The diterpene alkaloid 3-acetylaconitine showed significant antidepressant activity in a mouse behavioral despair model experiment, which was significantly better than the positive drug fluoxetine and can be used to develop antidepressant drugs.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and more specifically, relates to a C 19 A type diterpene alkaloid 3-acetylaconitine, a preparation method thereof, a pharmaceutical composition thereof and application thereof in antidepressant drugs. Background Art

[0002] In today's increasingly competitive society, high-intensity, high-stress lifestyles have a serious impact on people's physical and mental health. Depression is gradually increasing and seriously affecting people's normal work and life. Currently, drugs for treating depression generally have significant side effects and have certain limitations in clinical application.

[0003] The inventors prepared 3-acetylaconitine from the traditional Chinese medicine Aconite root and found that it showed significant antidepressant activity in a mouse behavioral despair model experiment, which was better than the positive drug Fluoxetine. It can be used to develop antidepressant drugs. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the present invention provides a C 19 A type diterpene alkaloid 3-acetylaconitine, a preparation method thereof, a pharmaceutical composition thereof and application thereof in antidepressant drugs.

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

[0006] A diterpene alkaloid 3-acetylaconitine having a structure shown in formula (1):

[0007] (1).

[0008] It should be noted that the diterpene alkaloid 3-acetylaconitine can be a compound, stereoisomer, tautomer, or pharmaceutically acceptable salt comprising the structure represented by formula (1). That is, the compound, stereoisomer, tautomer, and pharmaceutically acceptable salt thereof comprising the structure represented by formula (1) are all within the scope of protection of this application.

[0009] Another aspect of the present invention provides a method for preparing the diterpene alkaloid 3-acetylaconitine. The diterpene alkaloid 3-acetylaconitine is obtained by extraction and separation from aconite root.

[0010] In the above technical solution, the preparation method of the diterpene alkaloid 3-acetylaconitine comprises the following steps:

[0011] S1. Take dried aconite root, add solvent and reflux extract, combine the extracts and concentrate to obtain an extract;

[0012] S2, adding the extract to dilute hydrochloric acid to fully dissolve and suspend, filtering to remove impurities, and obtaining an acid aqueous solution; extracting the acid aqueous solution once with dichloromethane, filtering out the primary extract, and obtaining a filtered acid aqueous solution; adjusting the pH of the filtered acid aqueous solution to alkaline; and then performing a secondary extraction with dichloromethane to obtain a secondary extract;

[0013] S3. Gradient elution of the secondary extract with a dichloromethane-methanol solution was performed to obtain a fraction B; when the fraction B was identified by silica gel thin layer chromatography, the Rf value was 0.76-0.80;

[0014] S4, gradient eluting fraction B with a methanol-water mixed solution to obtain fraction B4;

[0015] When using silica gel thin layer chromatography, the Rf value corresponding to fraction B4 is 0.61-0.69;

[0016] S5. Gradient elution of fraction B4 with a methanol-water solution to obtain fraction L3;

[0017] When using silica gel thin layer chromatography, the Rf value corresponding to fraction L3 was 0.66-0.75;

[0018] S6. Separating the diterpene alkaloid 3-acetylaconitine from fraction L3 using RP-HPLC (i.e., reversed-phase high performance liquid chromatography);

[0019] The mobile phase used in the RP-HPLC method was an acetonitrile-water mixture.

[0020] Furthermore, in step S2, the filtered acid aqueous solution is adjusted to a pH of 8-11 with sodium hydroxide; preferably, the pH is adjusted to 9.

[0021] Furthermore, in step S2, the extract is added into 8-15 times the mass of dilute hydrochloric acid to be fully dissolved and suspended, and then filtered to remove impurities to obtain an acid aqueous solution.

[0022] Furthermore, the concentration of the dilute hydrochloric acid is 0.1-1.0%, preferably 0.5%.

[0023] Furthermore, in step S3, the secondary extract is separated by silica gel column chromatography to obtain fraction B; based on the total volume of 100, the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solution when fraction B is eluted (i.e., corresponding to fraction B) is (97:3)-(93:7).

[0024] Preferably, in step S3, the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solution when fraction B is washed out (i.e. corresponding to fraction B) is 95:5.

[0025] Furthermore, in step S4, the fraction B is separated by ODS column chromatography to obtain fraction B4; based on the total volume of 100, the volume ratio of methanol to water in the methanol-water mixed solution when fraction B4 is eluted (i.e., corresponding to fraction B4) is (45:55)-(55:45).

[0026] Preferably, in step S4, when fraction B4 is washed out, the volume ratio of methanol to water in the methanol-water mixed solution is 50:50.

[0027] Furthermore, in step S4, the methanol-water mixed solution contains 0.01-0.1V% formic acid.

[0028] Preferably, in step S4, the methanol-water mixed solution contains formic acid in an amount of 0.05 V%.

[0029] Furthermore, in step S5, the fraction B4 is separated by gel column chromatography to obtain fraction L3; based on the total volume of 100, the volume ratio of methanol to water in the methanol-water solution when fraction L3 is eluted (i.e., corresponding to fraction L3) is (65:35)-(75:25).

[0030] Preferably, in step S5, when fraction L3 is washed out, the volume ratio of methanol to water in the methanol-water solution is 70:30.

[0031] Furthermore, in step S6, the volume ratio of acetonitrile to water in the mobile phase acetonitrile-water mixed solution is (50:50)-(55:45); and the acetonitrile-water mixed solution contains 0.01-0.5V% trifluoroacetic acid.

[0032] Furthermore, in step S6, the volume ratio of acetonitrile to water in the mobile phase acetonitrile-water mixed solution is 52:48.

[0033] Furthermore, in step S6, the acetonitrile-water mixed solution contains 0.1V% trifluoroacetic acid.

[0034] Furthermore, in step S1, the solvent is an 88-98V% ethanol aqueous solution, the mass of the added solvent is 8-10 times that of the aconite root, the number of reflux extractions is 2-4 times, and each extraction takes 1-3 hours.

[0035] In another aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned diterpene alkaloid 3-acetylaconitine.

[0036] Furthermore, the pharmaceutical composition also includes a pharmacodynamically acceptable carrier or excipient.

[0037] Furthermore, the pharmaceutical composition further comprises a synergist;

[0038] Synergists are one or more of the following substances:

[0039] Fluoxetine, paroxetine, fluvoxamine, sertraline, citalopram, escitalopram, venlafaxine, duloxetine, mirtazapine, bupropion, agomelatine, trazodone, reboxetine, imipramine, amitriptyline, clomipramine, doxepin, maprotiline, moclobemide, Shugan Jieyu capsule, St. John's wort extract, flupentixol and melitracen, etc.

[0040] That is, the pharmaceutical composition containing the 3-acetylaconitine of the present invention as an active ingredient and conventional pharmaceutical excipients, adjuvants or carriers is also included in the present invention.

[0041] Furthermore, the dosage form of the pharmaceutical composition is tablets, capsules, granules, oral liquid, granules, pills or micropills.

[0042] In another aspect, the present invention further provides the use of the above-mentioned diterpene alkaloid 3-acetylaconitine and / or the pharmaceutical composition in antidepressant drugs.

[0043] Compared with the prior art, the present invention has the following advantages:

[0044] (1) The experimental results show that the C 19 The diterpene alkaloid 3-acetylaconitine showed significant antidepressant activity in the mouse behavioral despair model tail suspension test, which was superior to the positive drug fluoxetine and can be used to develop antidepressant drugs;

[0045] (2) The present invention provides C 19 The preparation method of the diterpene alkaloid 3-acetylaconitine can be obtained from aconite root with simple operation, good reproducibility and high extraction purity. 19 Method for preparing 3-acetylaconitine type diterpene alkaloid. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below with reference to the embodiments.

[0047] It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0048] In the examples, unless otherwise specified, all means used are conventional means in the art.

[0049] As used herein, the terms "comprise," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a listed element is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0050] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0051] The endpoints of the ranges and any values 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 endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0052] In addition, 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.

[0053] Example 1

[0054] This embodiment discloses a C 19 Preparation method of type diterpene alkaloid 3-acetylaconitine, the C 19 The diterpene alkaloid 3-acetylaconitine was extracted and isolated from Aconite root.

[0055] The C 19 The preparation method of the type diterpene alkaloid 3-acetylaconitine comprises the following steps:

[0056] S1. Take dried aconite root, add solvent and reflux extract, combine the extracts and concentrate to obtain an extract;

[0057] Specifically, dried aconite root and 95% ethanol aqueous solution with a mass 10 times that of the aconite root were taken as solvent, and refluxed for extraction three times, each extraction for 2 hours, and the extracts were combined and concentrated to obtain an extract;

[0058] S2. Add the extract to dilute hydrochloric acid, fully dissolve and suspend it, filter and remove impurities to obtain an acid aqueous solution; extract the acid aqueous solution once with dichloromethane, filter out the primary extract, and obtain a filtered acid aqueous solution; adjust the pH of the filtered acid aqueous solution to alkaline; and then perform a secondary extraction with dichloromethane to obtain a secondary extract.

[0059] Specifically, the extract was added to 10 times the mass of 0.5% dilute hydrochloric acid to fully dissolve and suspend, and then filtered to remove impurities. The filtered acid aqueous solution was extracted three times with 1.5 times the volume of dichloromethane to obtain an acidified dichloromethane extract (the first extract was discarded) and an acid aqueous layer, and the acid aqueous layer was the filtered acid aqueous solution;

[0060] The filtered acid aqueous solution was adjusted to pH = 9 with sodium hydroxide, and then extracted twice with dichloromethane.

[0061] S3. Gradient elution of the secondary extract was performed using a dichloromethane-methanol solution to obtain fraction B; when identified by silica gel thin layer chromatography, the brick-red spots indicated by the potassium bismuth iodide reagent corresponding to fraction B had an Rf value of 0.76-0.80; based on a total volume of 100, the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solution when fraction B was eluted (i.e., corresponding to fraction B) was (97:3)-(93:7).

[0062] Specifically, the secondary extract was chromatographed on a silica gel column using a gradient elution of dichloromethane-methanol with a volume ratio of 100:0, 95:5, 92:8, 90:10, 85:15, 75:25, 65:35, 50:50, and 0:100, respectively, to obtain fractions 1-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, and 81-90, for a total of 90 fractions.

[0063] Silica gel thin layer chromatography (TLC) was used to identify the brick-red spots revealed by potassium bismuth iodide reagent. The Rf values were 0.82-0.86 (fractions 1-10), 0.76-0.80 (fractions 11-20), 0.72-0.75 (fractions 21-30), 0.65-0.70 (fractions 31-40), 0.56-0.62 (fractions 41-50), 0.50-0.54 (fractions 51-60), 0.42-0.48 (fractions 61-70), 0.30-0.38 (fractions 71-80), and 0.25-0.28 (fractions 81-90). Similar fractions were combined to obtain fractions A, B, C, D, E, F, G, H, and I, respectively. Fractions 11-20 were combined to obtain fraction B.

[0064] Fraction B was gradient eluted with a methanol-water mixture to obtain Fraction B4. Fraction B4 exhibited an Rf value of 0.61-0.69 using silica gel thin-layer chromatography. Based on a total volume of 100, the volume ratio of methanol to water in the methanol-water mixture at the time of elution (i.e., corresponding to Fraction B4) was (45:55) to (55:45). The methanol-water mixture contained 0.01-0.1% by volume of formic acid.

[0065] That is, according to the brick red spots revealed by potassium bismuth iodide reagent and the characteristic ultraviolet absorption (λ max = 230 nm) and select fraction B for the next separation.

[0066] Specifically, fraction B was subjected to ODS column chromatography using a gradient elution using a methanol-water mixture (containing 0.05% formic acid in a methanol-water mixture) at a volume ratio of 20:80, 30:70, 40:60, 50:50, 60:40, 80:20, and 100:0. Fractions 1-6, 7-15, 16-23, 24-36, 37-45, 46-57, and 58-68 were obtained, for a total of 68 fractions.

[0067] Using silica gel thin-layer chromatography, the Rf values observed based on the brick-red spots revealed by potassium bismuth iodide reagent were 0.35-0.44 (fractions 1-6), 0.45-0.49 (fractions 7-15), 0.50-0.60 (fractions 16-23), 0.61-0.69 (fractions 24-36), 0.70-0.74 (fractions 37-45), 0.75-0.79 (fractions 46-57), and 0.80-0.89 (fractions 58-68). Similar fractions were combined to form seven fractions, B1 to B7. Fractions 24-36 were combined to obtain fraction B4.

[0068] S5. Fraction B4 was gradient eluted with a methanol-water solution to obtain fraction L3. When detected by silica gel thin layer chromatography, the Rf value of fraction L3 was 0.66-0.75. When fraction L3 was eluted, the volume ratio of methanol to water in the methanol-water solution was (65:35)-(75:25).

[0069] According to the brick-red spots revealed by potassium bismuth iodide reagent and the characteristic ultraviolet absorption (λ max = 230 nm) and fraction B4 was selected for the next separation.

[0070] The specific operation included subjecting fraction B4 to gel column chromatography using a methanol-water gradient elution ratio of 0:100, 30:70, 70:30, and 100:0 by volume, collecting fractions 1-6, 7-15, 16-21, and 22-25 in sequence. After 25 fractions were collected, they were identified by silica gel thin-layer chromatography. Based on the brick-red spots revealed by potassium bismuth iodide reagent, the Rf values were 0.35-0.52 (fractions 1-6), 0.53-0.65 (fractions 7-15), 0.66-0.75 (fractions 16-21), and 0.75-0.85 (fractions 22-25). Similar fractions were combined to form four fractions, L1 to L4. Fractions 16-21 were combined to obtain fraction L3. The Rf value of fraction L3 was 0.66-0.75.

[0071] According to the brick-red spots revealed by potassium bismuth iodide reagent and the characteristic ultraviolet absorption (λ max =230nm) and fraction L3 was selected for the next separation.

[0072] S6. Using RP-HPLC method, the diterpene alkaloid 3-acetylaconitine was separated from fraction L3 and defined as compound 1.

[0073] The mobile phase used in the RP-HPLC method is an acetonitrile-water mixed solution; the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is (50:50)-(55:45), preferably 52:48; wherein the acetonitrile-water mixed solution contains 0.01-0.5V% trifluoroacetic acid.

[0074] Specifically, the mobile phase used in the separation of fraction L3 by RP-HPLC was acetonitrile and water in a volume ratio of 52:48, and contained 0.1% trifluoroacetic acid; 18 The chromatographic column was prepared in fraction L3 to obtain C 19 Type diterpene alkaloid 3-acetylaconitine ( t R =28.2min, purity 98%).

[0075] The physical properties and test data of Compound 1 prepared in Example 1 are as follows:

[0076] White amorphous powder, easily soluble in methanol. High resolution mass spectrometry (HR-ESI-MS) m / z 688.3335 ([M+H] + The calculated value is 688.3328), and combined with the NMR data, the molecular formula is determined to be C 36 H 49 NO 12 , its unsaturation is calculated to be 13, and the NMR data are as follows:

[0077] 1 H NMR (400MHz, CDCl3) δ : 3.09 (1H, dd, J =10.6, 6.8Hz,H-1), 2.09 (1H, m,H-2β), 2.34 (1H, m, H-2α), 4.91 (1H, dd, J =13.8, 5.3Hz, H-3β), 2. 26 (1H, m,H-5β), 4.07 (1H, d, J =6.8 Hz, H-6β), 2.73 (1H, brs, H-7), 2.70 (1H, m, H-9), 2.21 (1H, m, H-10), 2.12 (1H, m, H-12α), 2.89 (1H, dd, J=12.2, 5.7Hz, H-12β),4.86 (1H, d, J =5.2 Hz, H-14β), 4.45 (1H, dd, J =5.2, 2.7Hz, H-15β), 3.31 (1H, d, J =5.3Hz, H-16), 2.82 (1H, s, H-17), 4.05 (1H, d, J =8.0Hz, H-18β), 3.79 (1H, d, J =8.4Hz, H-18α), 2.27 (1H, d, J =11.0 Hz, H-19β), 2.49 (1H, d, J =11.0 Hz, H-19α), 1.09 (3H, t, J =7.1Hz, NCH2CH3), 2.43 (1H, m, NCH2αCH3), 2.95 (1H, m, NCH2βCH3), 1.36 (3H, s, 8-COCH3), 2.05 (3H, s, 3-COCH3), 3.18 (3H, s, 1-OCH3), 3.17(3H, s, 6-OCH3), 3.71 (3H, s, 16-OCH3), 3.23 (3H, s, 18-OCH3), 8.04 (2H, d, J =7.1 Hz, H-2', 6'), 7.55 (1H, t, J =7.3 Hz, H-4'), 7.44 (2H, t, J =7.5 Hz, H-3',5');

[0078] 13 C NMR (100 MHz, CDCl3) δ: 83.7 (C-1), 32.2 (C-2), 71.9 (C-3), 42.5(C-4), 46.1 (C-5), 82.4 (C-6), 45.5 (C-7), 92.2 (C-8), 44.8 (C-9), 40.7 (C-10), 49.9 (C-11), 36.7 (C-12), 74.5 (C-13), 79.1 (C-14), 78.9 (C-15), 90.4(C-16), 61.4 (C-17), 71.7 (C-18), 49.4 (C-19), 47.3 (NCH2CH3), 13.7 (NCH2CH3),172.6 (8-COCH3), 21.4 (8-COCH3), 170.5 (3-COCH3), 21.6 (3-COCH3), 56.7 (1-OCH3), 58.6 (6-OCH3), 60.9 (16-OCH3), 58.9 (18-OCH3), 166.3 (OCO), 130.0 (C-1'), 129.8 (C-2', 6'), 128.8 (C-3', 5'), 133.5 (C-4').

[0079] The C 19 The structural formula of the diterpene alkaloid 3-acetylaconitine is shown below:

[0080]

[0081] (1).

[0082] Comparative Example 1

[0083] Comparative Example 1 of the present invention provides C 19 The preparation method of the diterpene alkaloid 3-acetylaconitine is similar to that of Example 1, except that in step S6, the mobile phase used for separating fraction L3 by RP-HPLC does not contain trifluoroacetic acid.

[0084] The compound prepared in Comparative Example 1 had severe peak tailing during the preparation process. Liquid phase analysis after preparation revealed that it contained a large amount of impurities, and the purity of the prepared compound 1 was less than 50%.

[0085] Comparative Example 2

[0086] Comparative Example 2 of the present invention provides C 19The preparation method of the diterpene alkaloid acetylaconitine is similar to that of Example 1, except that in step S4, when fraction B is subjected to ODS column chromatography, methanol-water (without formic acid) with an initial volume ratio of 20:80 is used for elution.

[0087] In Comparative Example 2, the compound could not be detected during the subsequent fraction preparation process;

[0088] The results showed that compound 1 could not be prepared.

[0089] Test Example 1

[0090] This test example discloses the antidepressant effect of the compound 3-acetylaconitine in a mouse behavioral despair model experiment (the gold standard for antidepressant drug screening).

[0091] 1. Experimental materials and instruments

[0092] Fluoxetine hydrochloride was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; methyltaurine and higenamine were purchased from Shanghai Shidande Standard Technology Service Co., Ltd.; total alkaloids from aconite root; 3-acetylaconitine.

[0093] ZIL-2 mouse self-activity box (Shanghai Xinman Science and Education Equipment Co., Ltd.); YLS-18A mouse tail suspension apparatus (Anhui Zhenghua Biological Instrument Equipment Co., Ltd.); electronic balance (Sartorius Scientific Instrument (Beijing) Co., Ltd.).

[0094] 2. Experimental Animals

[0095] Male ICR mice, weighing 18-22 g, were SPF-free and provided by Weitonglihua (Animal License No. SYXK (Beijing) 2023-0001). The animals were housed in an environment with a temperature of 23 ± 2°C and a humidity of 50 ± 10%, with a 12-hour light cycle (lights on from 7:00 AM to 7:00 PM) daily. Experiments were performed after 3 days of maintenance. The animals were fasted for 6 hours before behavioral experiments and had free access to water.

[0096] Experimental Methods: Seventy ICR male mice were screened and divided into seven groups, arranged in a serpentine pattern, based on the number of locomotor activity (locomotor activity) in descending order. These groups consisted of a blank control group (same volume of saline), a positive drug group (10 mg / kg) of fluoxetine, a methyltaurine group (10 mg / kg), a higenamine group (10 mg / kg), a group containing total aconite alkaloids (10 mg / kg), a low-dose 3-acetylaconitine group (0.03 mg / kg), and a high-dose 3-acetylaconitine group (0.3 mg / kg). Each group had 10 mice in each group and were numbered accordingly. All drugs were added to saline to form a solution. Each group was administered once daily by gavage at a volume of 0.1 ml / 10 g body weight. After three consecutive days of drug administration, locomotor activity was measured on the fourth day, and a tail suspension test was performed on the fifth day.

[0097] 3. Experimental results

[0098] As shown in Table 1, compared with the blank group, the high-dose group of 3-acetylaconitine (0.3 mg / kg) can significantly shorten the immobility time of mice in the tail suspension test, showing an antidepressant effect that is significantly better than the positive drugs fluoxetine (10 mg / kg, a first-line chemical drug for the clinical treatment of depression), methyltaurine (10 mg / kg), higenamine (10 mg / kg) and total alkaloids of aconite (10 mg / kg).

[0099] Table 1 Effects of 3-acetylaconitine on the tail suspension immobility time in behavioral despair model mice

[0100]

[0101] (Compared with the blank group, * P <0.05, *** P <0.001)

[0102] As shown in Table 2, compared with the blank group, each drug-treated group had no significant effect on the locomotor activity of mice in the open field test, indicating that the effect of each drug-treated group on the immobility time of mice in the tail suspension test was not related to neural excitability.

[0103] Table 2 Effects of 3-acetylaconitine on horizontal movement distance in the open field in behavioral despair model mice

[0104]

[0105] In summary, the C 19 The diterpene alkaloid 3-acetylaconitine has antidepressant activity that is significantly superior to fluoxetine, a first-line drug for depression, and can be used as a prodrug for the treatment of depression.

[0106] Application Example 1

[0107] The application example of the present invention discloses a capsule preparation using 3-acetylaconitine as a raw material drug, and its components are as follows:

[0108] 3-Acetylaconitine 3.5 mg

[0109] 6.0g starch

[0110] Sodium metabisulfite 0.2g

[0111] Magnesium stearate 0.2g

[0112] Anhydrous ethanol appropriate amount

[0113] Makes 100 tablets.

[0114] The specific preparation process is as follows:

[0115] 3-acetylaconitine, starch and sodium metabisulfite are mixed evenly, anhydrous ethanol is added to form a soft material, the mixture is passed through a 24-mesh sieve, formed into granules, dried, magnesium stearate is added, mixed evenly and put into capsules.

[0116] Application Example 2

[0117] The present invention discloses an application example of a granule preparation using the compound 3-acetylaconitine as a raw material drug, and its components are as follows:

[0118] 3-Acetylaconitine 4.0 mg

[0119] 6.0g starch

[0120] Sodium bisulfite 0.2g

[0121] Magnesium stearate 0.2g

[0122] Anhydrous ethanol appropriate amount

[0123] Makes 100 bags.

[0124] The specific preparation process is as follows:

[0125] 3-acetylaconitine is mixed with starch and sodium bisulfite, and then anhydrous ethanol is added to prepare a soft material. The mixture is passed through a 24-mesh sieve to prepare granules, dried, and magnesium stearate is added. The mixture is mixed and bagged.

[0126] Application Example 3

[0127] The application example of the present invention discloses an oral solution using the compound 3-acetylaconitine as a raw material drug, and its components are as follows:

[0128] 3-Acetylaconitine 6.0 mg

[0129] 3.0g sucrose

[0130] Sodium bisulfite 0.2g

[0131] Methyl parahydroxybenzoate 0.2g

[0132] Sodium bicarbonate 0.5g

[0133] Water for injection 1000.0 mL

[0134] Made into 100 pieces.

[0135] The specific preparation process is as follows:

[0136] After the above components are mixed evenly, they can be packaged using a conventional oral liquid preparation method.

[0137] Application Example 4

[0138] The application example of the present invention discloses an injection with the compound 3-acetylaconitine as the raw material drug, and its components are as follows:

[0139] 3-Acetylaconitine 4.0 mg

[0140] Vitamin C 0.2g

[0141] Sodium chloride 6.0g

[0142] Sodium bicarbonate 0.5g

[0143] Water for injection 1000.0 mL

[0144] Made into 100 pieces.

[0145] The specific preparation process is as follows:

[0146] After the above components are mixed evenly, 100 injections can be obtained using conventional injection preparation methods.

[0147] Application Example 5

[0148] The application example of the present invention discloses a tablet with the compounds 3-acetylaconitine and fluoxetine as raw materials, and its components are as follows:

[0149] 3-Acetylaconitine 3.5 mg

[0150] Fluoxetine 20mg

[0151] Hydroxypropyl methylcellulose 18g

[0152] 0.4g talcum powder

[0153] Lactose 0.2g

[0154] Magnesium stearate 0.2g

[0155] Anhydrous ethanol appropriate amount

[0156] Made into 100 pieces.

[0157] The specific preparation process is as follows:

[0158] 3-acetylaconitine, fluoxetine, hydroxypropyl methylcellulose, talc, lactose and magnesium stearate are mixed evenly, anhydrous ethanol is added to form a soft material, the material is passed through a 24-mesh sieve to form granules, dried, magnesium stearate is added, mixed evenly and tableted.

[0159] Application Example 6

[0160] The application example of the present invention discloses a capsule preparation using the compound 3-acetylaconitine and Luyoutai as raw materials, and its components are as follows:

[0161] 3-Acetylaconitine 3.5 mg

[0162] Luyoutai 300.0mg

[0163] 6.0g starch

[0164] Sodium metabisulfite 0.2g

[0165] Magnesium stearate 0.2g

[0166] Anhydrous ethanol appropriate amount

[0167] Makes 100 tablets.

[0168] The specific preparation process is as follows:

[0169] Take 3-acetylaconitine, Luyoutai and sodium metabisulfite and mix them evenly, add anhydrous ethanol to make a soft material, pass through a 24-mesh sieve, make granules, dry, add magnesium stearate, mix evenly, and put into capsules.

[0170] Application Example 7

[0171] The application example of the present invention discloses an injection with the compound 3-acetylaconitine and paroxetine as raw materials, and its components are as follows:

[0172] 3-Acetylaconitine 5.0 mg

[0173] Paroxetine 20.0mg

[0174] Vitamin C 0.2g

[0175] Sodium chloride 6.0g

[0176] Sodium bicarbonate 0.5g

[0177] Water for injection 1000.0 mL;

[0178] Made into 100 pieces.

[0179] The specific preparation process is as follows:

[0180] After the above components are mixed evenly, 100 injections can be obtained using conventional injection preparation methods.

[0181] The above-mentioned embodiments only express several implementation methods of the present invention. The description thereof is relatively specific and detailed, but it should not be understood as limiting the scope of the invention patent.

[0182] It should be noted that those skilled in the art may make various modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims.

Claims

1. A method for preparing a diterpene alkaloid 3-acetylaconitine, characterized in that: The diterpene alkaloid 3-acetylaconitine has a structure shown in formula (1): The diterpene alkaloid 3-acetylaconitine is extracted and separated from Aconite root; The preparation method comprises the following steps: S1. Take dried aconite root, add solvent and reflux extract, combine the extracts and concentrate to obtain an extract; S2, adding the extract to dilute hydrochloric acid to fully dissolve and suspend, filtering to remove impurities, and obtaining an acid aqueous solution; extracting the acid aqueous solution once with dichloromethane, filtering out the primary extract, and obtaining a filtered acid aqueous solution; adjusting the pH of the filtered acid aqueous solution to alkaline; and then performing a secondary extraction with dichloromethane to obtain a secondary extract; S3. Gradient elution of the secondary extract with a dichloromethane-methanol solution was performed to obtain a fraction B; when the fraction B was identified by silica gel thin layer chromatography, the Rf value was 0.76-0.80; The secondary extract is separated by silica gel column chromatography to obtain fraction B; based on the total volume being 100, the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solution when eluting fraction B is (97:3)-(93:7); S4, gradient eluting fraction B with a methanol-water mixed solution to obtain fraction B4; When using silica gel thin layer chromatography, the Rf value corresponding to fraction B4 is 0.61-0.69; Fraction B is separated by ODS column chromatography to obtain fraction B4; based on the total volume being 100, the volume ratio of methanol to water in the methanol-water mixed solution when fraction B4 is eluted is (45:55)-(55:45); The methanol-water mixture contains 0.01-0.1V% formic acid; S5. Gradient elution of fraction B4 with a methanol-water solution to obtain fraction L3; When using silica gel thin layer chromatography, the Rf value corresponding to fraction L3 was 0.66-0.75; Fraction B4 is separated by gel column chromatography to obtain fraction L3; based on the total volume being 100, the volume ratio of methanol to water in the methanol-water solution when fraction L3 is eluted is (65:35)-(75:25); S6. Separating the diterpene alkaloid 3-acetylaconitine from fraction L3 using RP-HPLC; The mobile phase used in the RP-HPLC method is an acetonitrile-water mixed solution; the volume ratio of acetonitrile to water in the mobile phase acetonitrile-water mixed solution is (50:50)-(55:45); and the acetonitrile-water mixed solution contains 0.01-0.5V% trifluoroacetic acid.

2. The preparation method according to claim 1, characterized in that In step S2, the filtered acid aqueous solution is adjusted to a pH of 8-11 with sodium hydroxide.

3. The preparation method according to claim 1, characterized in that In step S2, the extract is added into 8-15 times the mass of dilute hydrochloric acid to fully dissolve and suspend, and then filtered to remove impurities to obtain an acid aqueous solution.

4. The preparation method according to claim 1, characterized in that The concentration of the dilute hydrochloric acid is 0.1-1.0%.

5. The preparation method according to claim 1, characterized in that In step S3, the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solution when fraction B is eluted is 95:

5.

6. The preparation method according to claim 1, characterized in that In step S4, when fraction B4 is washed out, the volume ratio of methanol to water in the methanol-water mixed solution is 50:

50.

7. The preparation method according to claim 1, characterized in that In step S4, the volume of formic acid contained in the methanol-water mixed solution is 0.05V%.

8. The preparation method according to claim 1, characterized in that In step S5 , when fraction L3 is washed out, the volume ratio of methanol to water in the methanol-water solution is 70:

30.

9. The preparation method according to claim 1, characterized in that In step S6, the volume ratio of acetonitrile to water in the mobile phase acetonitrile-water mixed solution is 52:

48.

10. The preparation method according to claim 1, characterized in that In step S6, the acetonitrile-water mixed solution contains 0.1V% trifluoroacetic acid.

11. The preparation method according to claim 1, characterized in that In step S1, the solvent is 88-98V% ethanol aqueous solution, the mass of the added solvent is 8-10 times that of the aconite root, the number of reflux extractions is 2-4 times, and each extraction takes 1-3 hours.

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