Lappaconitine and its preparation method, pharmaceutical composition and application in antidepressant drugs

Abstract

The present invention relates to the field of pharmaceutical technologies, and discloses lappaconitine, a preparation method thereof, a pharmaceutical composition, and an application in antidepressant drugs; among them, the lappaconitine, a type C 18 diterpenoid alkaloid provided by the present invention shows significant antidepressant activity in the mouse behavioral despair model experiment, is significantly superior to the positive drug fluoxetine, and can be used for developing antidepressant drugs.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and more specifically, relates to a C 18 type diterpenoid alkaloid compound, lappaconitine, its preparation method, pharmaceutical composition and application in antidepressant drugs. Background Art

[0002] Nowadays, social competition is becoming increasingly fierce. The high-intensity and high-pressure life has a serious impact on people's physical and mental health. Depression is gradually increasing and seriously affects people's normal work and life. At present, the drugs for treating depression generally have relatively large side effects and have certain limitations in clinical applications.

[0003] As a first-in-class non-addictive analgesic drug in China, lappaconitine has the advantages of good analgesic activity, long-lasting effect and few adverse reactions. It is widely used clinically in the treatment of mild to moderate pain such as postoperative pain and cancer pain. At present, there are also related studies on the pharmacological activities of lappaconitine such as anti-arrhythmia, anti-inflammatory and anti-tumor, but there is no report on its antidepressant activity.

[0004] The inventor prepared lappaconitine from the traditional Chinese medicine Aconitum carmichaelii Debx. and found that it showed significant antidepressant activity in the mouse behavioral despair model experiment, superior to the positive drug fluoxetine, and can be used for the development of antidepressant drugs. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, the present invention provides a preparation method, pharmaceutical composition and application in antidepressant drugs of a C 18 type diterpenoid alkaloid, lappaconitine.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] A lappaconitine having the structure shown in formula (1):

[0008]

[0009] It should be noted that the lappaconitine may be a compound, stereoisomer, tautomer or pharmaceutically acceptable salt including the structure shown in formula (1). That is, the compound including the structure shown in formula (1), its stereoisomers, tautomers and pharmaceutically acceptable salts are all within the protection scope of the present application.

[0010] Another aspect of the present invention also provides a preparation method of the above lappaconitine, and the lappaconitine is obtained by extraction and separation from Aconitum carmichaelii Debx.

[0011] In the above technical solution, the preparation method of lappaconitine includes the following steps:

[0012] S1. Take dried Aconitum carmichaelii Debx., add a solvent for reflux extraction, combine the extraction solutions and concentrate to obtain an extract.

[0013] S2. Add the extract to dilute hydrochloric acid, fully dissolve and suspend it, then filter to remove impurities to obtain an acidic aqueous solution; perform a single extraction on the acidic aqueous solution with dichloromethane, filter off the single extract to obtain a filtered acidic aqueous solution; adjust the pH of the filtered acidic aqueous solution to alkaline; then perform a second extraction with dichloromethane to obtain a second extract.

[0014] S3. Perform gradient elution on the second extract with a dichloromethane-methanol solution to obtain fraction B; when using silica gel thin layer chromatography for identification, the Rf value corresponding to fraction B is 0.76 - 0.80.

[0015] S4. Perform gradient elution on fraction B with a methanol-water mixed solution to obtain fraction B2.

[0016] When using silica gel thin layer chromatography for identification, the Rf value corresponding to fraction B2 is 0.45 - 0.49.

[0017] S5. Perform gradient elution on fraction B2 with a methanol-aqueous solution to obtain fraction K3.

[0018] When using silica gel thin layer chromatography for identification, the K3 value corresponding to fraction B2 is 0.64 - 0.76.

[0019] S6. Separate the lappaconitine from fraction K3 by RP-HPLC method (i.e., reverse phase high performance liquid chromatography separation method); the mobile phase used in the RP-HPLC method is an acetonitrile-water mixed solution.

[0020] Further, in step S2, use sodium hydroxide to adjust the pH of the filtered acidic aqueous solution to 8 - 11; preferably, pH = 9.

[0021] Further, in step S2, add the extract to 8 - 15 times the mass of dilute hydrochloric acid, fully dissolve and suspend it, then filter to remove impurities to obtain an acidic aqueous solution.

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

[0023] Further, in step S3, the second extract is separated by silica gel column chromatography to obtain fraction B; based on a total volume of 100 as a reference, the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solution when eluting fraction B (i.e., the dichloromethane-methanol solution corresponding to fraction B) is (97:3) - (93:7); preferably, the volume ratio of dichloromethane to methanol is 95:5.

[0024] Further, in step S4, fraction B is separated by ODS column chromatography to obtain fraction B2; based on a total volume of 100, the volume ratio of methanol to water in the methanol-water mixed solution when eluting fraction B2 (i.e., corresponding to fraction B2) is (25:75)-(35:65); preferably, the volume ratio of methanol to water is 30:70.

[0025] Further, in step S4, the methanol-water mixed solution contains 0.01-1.1 V% formic acid.

[0026] Preferably, in step S4, the methanol-water mixed solution contains 0.05 V% formic acid.

[0027] Further, in step S5, fraction B2 is separated by gel column chromatography to obtain fraction K3; based on a total volume of 100, the volume ratio of methanol to water in the methanol-water solution when eluting fraction K3 (i.e., corresponding to fraction K3) is (65:35)-(75:25); preferably, the volume ratio of methanol to water is 70:30.

[0028] Preferably, the gel column chromatography is Sephadex LH-20 gel column chromatography.

[0029] Further, in step S6, the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is (44:56)-(48:52); and the acetonitrile-water mixed solution contains 0.01-0.5 V% trifluoroacetic acid.

[0030] Preferably, in step S6, the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is 46:54.

[0031] Preferably, in step S6, the acetonitrile-water mixed solution contains 0.1 V% trifluoroacetic acid.

[0032] Further, in step S1, the solvent is an ethanol aqueous solution of 88-98 V%, the mass of the solvent added is 8-10 times that of the aconite root, the number of times of reflux extraction is 2-4 times, and each extraction is for 1-3 h.

[0033] Another aspect of the present invention also provides a pharmaceutical composition, which comprises the above-mentioned lappaconitine.

[0034] Further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0035] Further, the pharmaceutical composition further comprises a synergist;

[0036] The synergist is one or several of the following substances:

[0037] Fluoxetine, paroxetine, fluvoxamine, sertraline, citalopram, escitalopram, venlafaxine, duloxetine, mirtazapine, bupropion, agomelatine, trazodone, reboxetine, imipramine, amitriptyline, clomipramine, doxepin, maprotiline, moclobemide, Shugan Jieyu Capsule, Hypericum perforatum extract, flupentixol melitracen, etc.

[0038] That is, a pharmaceutical composition containing lappaconitine of the present invention as an active ingredient and a conventional pharmaceutical excipient or adjuvant or carrier is also included in the present invention.

[0039] Furthermore, the dosage form of the pharmaceutical composition is tablets, capsules, granules, oral liquids, instant granules, dripping pills or pellets.

[0040] Another aspect of the present invention also provides the use of the above-mentioned lappaconitine or the above-mentioned pharmaceutical composition in antidepressant drugs.

[0041] Compared with the prior art, the present invention has the following advantages:

[0042] (1) Experimental results show that the lappaconitine provided by the present invention exhibits significant antidepressant activity in the tail suspension test of the mouse behavioral despair model, which is superior to the positive drug fluoxetine and can be used for developing antidepressant drugs;

[0043] (2) The present invention provides a method for extracting lappaconitine from Aconitum carmichaelii Debx. with simple operation, good reproducibility and high extraction purity. Detailed implementation manners

[0044] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following further describes the present invention in detail with reference to embodiments.

[0045] It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0046] In the embodiments, unless otherwise specified, the means used are all conventional means in the art.

[0047] As used herein, the term "comprising", "including" or any other variation thereof is intended to cover a non-exclusive inclusion. For example, a composition, step, method, article or device containing 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 device.

[0048] For those not specifying specific techniques or conditions in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. For those reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0049] The endpoints and any values within the ranges disclosed herein are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered as specifically disclosed herein.

[0050] 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.

[0051] Example 1

[0052] A method for preparing lappaconitine, a type C 18 diterpenoid alkaloid, comprises the following steps:

[0053] S1. Take dried Aconiti Lateralis Radix Praeparata, add a solvent for reflux extraction, and concentrate the combined extraction solution to obtain an extract. Specifically, take 200.5 kg of dried Aconiti Lateralis Radix Praeparata, and use an aqueous ethanol solution of 95% with a volume 10 times that of Aconiti Lateralis Radix Praeparata as the solvent for reflux extraction three times, each extraction for 2 h. After concentrating the combined extraction solution, 6 kg of extract is obtained.

[0054] S2. Add the extract to dilute hydrochloric acid, fully dissolve and suspend it, then filter to remove impurities to obtain an acidic aqueous solution. Extract the acidic aqueous solution once with dichloromethane, filter off the first extractant, and obtain a filtered acidic aqueous solution. Adjust the pH of the filtered acidic aqueous solution to alkaline, and then perform a second extraction with dichloromethane to obtain a second extractant.

[0055] In this example, add the extract to 0.5% dilute hydrochloric acid (60 L) with a mass 10 times that of the extract, fully dissolve and suspend it, then filter to remove impurities to obtain an acidic aqueous solution. Extract the acidic aqueous solution 3 times with 1.5 times the volume of dichloromethane to obtain 2.5 kg of the first acidified dichloromethane extract (discarded) and 1.2 kg of the filtered acidic aqueous solution, i.e., the acid aqueous layer. Adjust the filtered acidic aqueous solution to pH = 9 with sodium hydroxide, and then still extract with dichloromethane. The second dichloromethane extractant is 500 g this time.

[0056] S3. Perform gradient elution on the second extractant with a dichloromethane - methanol solution to obtain fraction B. When using silica gel thin layer chromatography for identification, the Rf value corresponding to fraction B is 0.76 - 0.80.

[0057] By silica gel column chromatography, the secondary extract was gradient eluted with dichloromethane-methanol with volume ratios of 100:0, 95:5, 92:8, 90:10, 85:15, 75:25, 65:35, 50:50, 0:100 in turn to obtain fractions 1-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90. After collecting 90 fractions in total, silica gel thin layer chromatography was used for identification. According to the brick red spots shown by Dragendorff's reagent, the Rf values were observed to be 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), 0.25-0.28 (fractions 81-90). Similar fractions were combined to obtain fractions A, B, C, D, E, F, G, H and I in turn; among them, fractions 11-20 were combined to obtain fraction B.

[0058] According to the brick red spots shown by Dragendorff's reagent and the characteristic ultraviolet absorption of diterpenoid alkaloids observed by HPLC analysis (λ max = 230 nm), fraction B was selected for the next separation.

[0059] S4. Fraction B was gradient eluted with a methanol-water mixed solution to obtain fraction B2; when silica gel thin layer chromatography was used for identification, the Rf value corresponding to fraction B2 was 0.45-0.49. When fraction B2 was eluted, the volume ratio of methanol to water in the methanol-water mixed solution was (25:75)-(35:65); preferably, the volume ratio of methanol to water was 30:70. In addition, the methanol-water mixed solution contained 0.01-1.1 V% formic acid, preferably 0.05 V% formic acid.

[0060] The specific operations include: subjecting fraction B to ODS column chromatography, and performing gradient elution with methanol-water (containing 0.05% formic acid) at volume ratios of 20:80, 30:70, 40:60, 50:50, 60:40, 80:20, and 100:0, successively obtaining fractions 1-6, 7-15, 16-23, 24-36, 37-45, 46-57, and 58-68. A total of 68 fractions are collected, and silica gel thin layer chromatography is used for identification. According to the brick red spots developed by Dragendorff's reagent, the Rf values are observed to be 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 are combined into 7 fractions B1 to B7. After combining fractions 7-15, fraction B2 is obtained.

[0061] Based on the brick red spots developed by Dragendorff's reagent and the characteristic ultraviolet absorption of diterpenoid alkaloids observed by HPLC analysis (λ max = 230 nm), fraction B2 is selected for the next separation step.

[0062] S5. Perform gradient elution on fraction B2 with a methanol-water solution to obtain fraction K3. When using silica gel thin layer chromatography for identification, the K3 value corresponding to fraction B2 is 0.64-0.76. The volume ratio of methanol to water in the methanol-water solution when eluting fraction K3 (i.e., the methanol-water solution corresponding to fraction K3) is (65:35)-(75:25); preferably, the volume ratio of methanol to water is 70:30.

[0063] The specific operations include: subjecting fraction B2 to Sephadex LH-20 gel column chromatography, and performing gradient elution with methanol-water at volume ratios of 0:100, 30:70, 70:30, and 100:0, successively collecting fractions 1-4, 5-13, 14-19, and 20-24. After a total of 24 fractions are collected, silica gel thin layer chromatography is used for identification. Based on the brick red spots developed by Dragendorff's reagent, the Rf values are observed to be 0.30-0.55 (fractions 1-4), 0.56-0.63 (fractions 5-13), 0.64-0.76 (fractions 14-19), and 0.77-0.85 (fractions 20-24). Similar fractions are combined into 4 fractions K1 to K4; after combining fractions 14-19, fraction K3 is obtained.

[0064] In this step, based on the brick red spots developed by Dragendorff's reagent and the characteristic ultraviolet absorption of diterpenoid alkaloids observed by HPLC analysis (λ max = 230 nm), fraction K3 is selected for the next separation step.

[0065] S6. The lappaconitine is separated from fraction K3 by RP-HPLC method; 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 (44:56)-(48:52); the acetonitrile-water mixed solution contains 0.01-0.5V% trifluoroacetic acid. Preferably, the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is 46:54, and the acetonitrile-water mixed solution contains 0.1V% trifluoroacetic acid.

[0066] The specific operation includes: using an acetonitrile-water mixed solution (containing 0.1% trifluoroacetic acid) as the mobile phase, adopting the HPLC method, with the volume ratio of acetonitrile to water being 46:54, and using a C 18 chromatographic column to prepare lappaconitine (t R = 18.5 min, purity 95%) from fraction K3, defined as compound 1.

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

[0068] White amorphous powder, soluble in methanol. High-resolution mass spectrometry (HR-ESI-MS) m / z 585.3160 ([M+H] + The calculated value is 585.3170), combined with NMR data, to determine its molecular formula as C 32 H 44 N2O8, calculate its degree of unsaturation as 12, and the NMR data are as follows:

[0069] 1 H NMR (400 MHz, CDCl3) δ: 11.04 (1H, s, NHCO3), 8.64 (1H, d, J = 8.4 Hz, H-6′, Ar-H), 7.91 (1H, dd, J = 8.0, 1.2 Hz, H-3′, Ar-H), 7.47 (1H, td, J = 8.0, 1.6 Hz, H-4′, Ar-H), 7.00 (1H, td, J = 8.0, 1.2 Hz, H-5′, Ar-H), 3.30, 3.31, 3.38 (each 3H, s, 3×OCH3), 2.20 (3H, s, COCH3), 1.11 (3H, t, J = 6.8 Hz, N-CH2CH3);

[0070] 1313C NMR (100 MHz, CDCl3) δ: 169.0 (NHCOCH3), 167.5 (ArCO), 141.7 (C-2′), 134.5 (C-4′), 131.1 (C-6′), 122.4 (C-5′), 120.3 (C-3′), 115.8 (C-1′), 90.2 (C-14), 84.7 (C-4), 84.3 (C-1), 82.9 (C-16), 78.5 (C-9), 75.7 (C-8), 61.6 (C-17), 57.8 (14-OCH3), 56.6 (1-OCH3), 56.2 (16-OCH3), 55.6 (C-19), 50.8 (C-11), 49.8 (C-10), 49.1 (C-21), 48.5 (C-5), 47.6 (C-7), 44.8 (C-15), 36.3 (C-13), 31.8 (C-3), 26.8 (C-6), 26.2 (C-2), 25.5 (NHCOCH3), 24.1 (C-12), 13.5 (C-22).

[0071] The said C 18 The structural formula of the diterpenoid alkaloid lappaconitine of type C is as shown in the following formula:

[0072]

[0073] Comparative Example 1

[0074] Comparative Example 1 of the present invention provides a preparation method of the diterpenoid alkaloid lappaconitine of type C. The steps are similar to those of Example 1, except that in step S6, in the mobile phase used for separating fraction K3 by RP-HPLC method, the aqueous solution of the mobile phase does not contain trifluoroacetic acid. 18 In the preparation process of the compound obtained in Comparative Example 1, the peak shape was severely tailed. After preparation, analysis by liquid chromatography detected a large amount of impurities. The purity of the prepared compound 1 was less than 50%.

[0075] In the preparation process of the compound obtained in Comparative Example 1, the peak shape was severely tailed. After preparation, analysis by liquid chromatography detected a large amount of impurities. The purity of the prepared compound 1 was less than 50%.

[0076] Comparative Example 2

[0077] Comparative Example 2 of the present invention provides a preparation method of the diterpenoid alkaloid lappaconitine of type C. The steps are similar to those of Example 1, except that in step S4, when fraction B was chromatographed on an ODS column, methanol-water (without formic acid) with an initial volume ratio of 20:80 was used for elution. In Comparative Example 2, the compound could not be detected during the subsequent fraction preparation process. The results showed that compound 1 could not be prepared. 18 In the subsequent fraction preparation process of Comparative Example 2, the compound could not be detected. The results showed that compound 1 could not be prepared.

[0078] Test Example 1

[0079] This experimental example discloses the antidepressant effect of the above-mentioned compound lappaconitine in the mouse behavioral despair model experiment (the gold standard for screening antidepressant drugs).

[0080] 1. Experimental materials and instruments

[0081] Fluoxetine hydrochloride was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; demethylcoclaurine and methyllycaconitine were purchased from Shanghai Standard Technology Service Co., Ltd.; total alkaloids of Aconitum carmichaelii Debx.; lappaconitine.

[0082] ZIL-2 type mouse locomotor activity box (Shanghai Xinman Science and Education Equipment Co., Ltd.);

[0083] YLS-18A type mouse tail suspension apparatus (Anhui Zhenghua Biological Instrument Equipment Co., Ltd.); electronic balance (Sartorius Scientific Instruments Co., Ltd., Beijing).

[0084] 2. Experimental animals

[0085] ICR mice, male, weighing 18 - 22 g, SPF grade, provided by Vital River Laboratory Animal Technology Co., Ltd., animal license number: SYXK(Beijing)2023 - 0001. The animals were housed in an environment with a temperature of 23 ± 2 °C and a humidity of 50% ± 10%, and the lighting time was 12 h per day (7:00 - 19:00 lighting). After 3 days of animal feeding, various experiments were carried out. 6 h before the start of the behavioral experiments, the animals were fasted and given free access to water.

[0086] 3. Experimental methods

[0087] 70 male ICR mice were arranged in a snake-like pattern and divided into 7 groups according to the descending order of the number of spontaneous activities during animal screening, namely the blank group (normal saline of the same volume), the positive drug fluoxetine hydrochloride group (10 mg / kg), the methyllycaconitine group (10 mg / kg), the demethylcoclaurine group (10 mg / kg), the total alkaloids of Aconitum carmichaelii Debx. group (10 mg / kg), the low-dose lappaconitine group (0.03 mg / kg), and the high-dose lappaconitine group (0.3 mg / kg); 10 mice in each group were numbered separately. The drugs were all added to normal saline to prepare a suspension. Each group was given intragastric administration once a day, and the administration volume was 0.1 ml / 10 g body weight. After continuous administration for 3 days, the locomotor activity experiment was carried out on the 4th day, and the tail suspension experiment was carried out on the 5th day.

[0088] 3. Experimental results

[0089] As shown in Table 1, compared with the blank group, the low and high dose groups of lappaconitine (0.03, 0.3 mg / kg) could significantly shorten the immobility time of mice in the tail suspension test, showing significant antidepressant activity, which was significantly better than the positive drugs fluoxetine (10 mg / kg, the first-line chemical drug for clinical treatment of depression), methyllycaconitine (10 mg / kg), demethylcoclaurine (10 mg / kg) and total alkaloids of Aconitum carmichaelii Debx. (10 mg / kg).

[0090] Table 1 Effects of lappaconitine on the immobility time of mice in the behavioral despair model

[0091]

[0092] (Mean ± standard deviation, compared with the blank group, *** P<0.001)

[0093] As shown in Table 2, compared with the blank group, each administration group had no significant effect on the spontaneous activity of mice in the open field test, indicating that the effect of each administration group on the immobility time of mice in the tail suspension test was not related to nerve excitability.

[0094] Table 2 Effects of lappaconitine on the horizontal movement distance of mice in the behavioral despair model

[0095]

[0096] In summary, the C 18 diterpenoid alkaloid lappaconitine of the present invention has antidepressant activity significantly superior to that of fluoxetine, the first-line drug for treating depression, and can be used as a drug precursor for the treatment of depression.

[0097] Application Example 1

[0098] The application example of the present invention discloses a capsule using lappaconitine as the raw material, and its components are as follows:

[0099]

[0100] The specific preparation process is as follows:

[0101] Take lappaconitine, starch and sodium metabisulfite, mix them evenly, add anhydrous ethanol to make soft materials, pass through a 24-mesh sieve, make granules, dry, add magnesium stearate, mix evenly, and fill into capsules.

[0102] Application Example 2

[0103] The application example of the present invention discloses a granule using the compound lappaconitine as the raw material, and its components are as follows:

[0104] Lappaconitine 4.0 mg

[0105] Starch 6.0 g

[0106] 0.2 g of sodium bisulfite

[0107] 0.2 g of magnesium stearate

[0108] An appropriate amount of absolute ethanol

[0109] Manufacture 100 bags.

[0110] The specific preparation process is as follows:

[0111] Take lappaconitine, starch and sodium bisulfite, mix them evenly, add absolute ethanol to make soft materials, pass through a 24-mesh sieve, make granules, dry them, add magnesium stearate, mix them evenly, and bag them.

[0112] Application Example 3

[0113] The application example of the present invention discloses an oral liquid with compound lappaconitine as the raw material medicine, and its components are as follows:

[0114]

[0115] The specific preparation process is as follows:

[0116] After mixing the above components, adopt the conventional preparation method of oral liquid and carry out sub-packaging.

[0117] Application Example 4

[0118] The application example of the present invention discloses an injection with compound lappaconitine as the raw material medicine respectively, and its components are as follows:

[0119]

[0120] The specific preparation process is as follows:

[0121] After mixing the above components, adopt the conventional preparation method of injection, and 100 injections can be obtained.

[0122] Application Example 5

[0123] The application example of the present invention discloses a tablet with compound lappaconitine and fluoxetine as the raw material medicines, and its components are as follows:

[0124]

[0125] The specific preparation process is as follows:

[0126] Take lappaconitine, fluoxetine, hydroxypropyl methylcellulose, talc, lactose and magnesium stearate, mix them evenly, add absolute ethanol to make soft materials, pass through a 24-mesh sieve, make granules, dry them, add magnesium stearate, mix them evenly, and press tablets.

[0127] Application Example 6

[0128] The application example of the present invention discloses a capsule using lappaconitine and hypericum perforatum as raw materials, and its components are as follows:

[0129]

[0130] The specific preparation process is as follows:

[0131] Take lappaconitine, hypericum perforatum, and sodium metabisulfite, mix them evenly, add anhydrous ethanol to make soft materials, pass through a 24-mesh sieve to make granules, dry them, add magnesium stearate, mix them evenly, and fill them into capsules.

[0132] Application Example 7

[0133] The application example of the present invention discloses an injection using lappaconitine and paroxetine as raw materials, and its components are as follows:

[0134]

[0135] The specific preparation process is as follows:

[0136] After mixing the above components, using the conventional preparation method for injections, 100 vials can be obtained.

[0137] The above embodiments only represent several implementation modes of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent.

[0138] It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A preparation method of lappaconitine, characterized in that, Lappaconitine has the structure shown in formula (1): （1）； The lappaconitine is obtained by extraction and separation from Aconitum carmichaelii Debx.; The preparation method comprises the following steps: S1. Take the dried Aconitum carmichaelii Debx., add a solvent for reflux extraction, combine the extraction solutions and concentrate to obtain an extract; S2. Add the extract to dilute hydrochloric acid for full dissolution and suspension, filter to remove impurities to obtain an acidic aqueous solution; perform a single extraction of the acidic aqueous solution with dichloromethane, filter off the single extract to obtain a filtered acidic aqueous solution; adjust the pH of the filtered acidic aqueous solution to be alkaline; then perform a secondary extraction with dichloromethane to obtain a secondary extract; S3. Perform gradient elution on the secondary extract with a dichloromethane-methanol solution to obtain fraction B; when detected by silica gel thin layer, the Rf value corresponding to fraction B is 0.76 - 0.80; S4. Perform gradient elution on fraction B with a methanol-water mixed solution to obtain fraction B2; when detected by silica gel thin layer, the Rf value corresponding to fraction B2 is 0.45 - 0.49; S5. Perform gradient elution on fraction B2 with a methanol-aqueous solution to obtain fraction K3; When detected by silica gel thin layer, the K3 value corresponding to fraction B2 is 0.64 - 0.76; S6. Separate the lappaconitine from fraction K3 by RP-HPLC method; The mobile phase used in the RP-HPLC method is an acetonitrile-water mixed solution.

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

3. The preparation method according to claim 1, wherein, In step S2, the pH of the filtered acidic aqueous solution is adjusted to 9 with sodium hydroxide.

4. The preparation method according to claim 1, characterized in that, In step S2, add the extract to 8 - 15 times the mass of dilute hydrochloric acid for full dissolution and suspension, filter to remove impurities to obtain an acidic aqueous solution.

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

6. The preparation method according to claim 1, wherein, In step S3, the secondary extract is separated by silica gel column chromatography to obtain fraction B; based on a total volume of 100, the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solution when eluting fraction B is (97:3) - (93:7).

7. The preparation method according to claim 6, characterized in that, The volume ratio of dichloromethane to methanol in the dichloromethane-methanol solution when eluting fraction B is 95:

5.

8. The preparation method according to claim 1, characterized in that, In step S4, fraction B is separated by ODS column chromatography to obtain fraction B2; based on a total volume of 100, the volume ratio of methanol to water in the methanol-water mixed solution when eluting fraction B2 is (25:75) - (35:65).

9. The preparation method according to claim 8, characterized in that, In step S4, the volume ratio of methanol to water in the methanol-water mixed solution when eluting fraction B2 is 30:

70.

10. The preparation method according to claim 1, characterized in that, In step S4, the methanol-water mixed solution contains 0.01 - 1.1V% formic acid.

11. The preparation method according to claim 10, characterized in that, In step S4, the methanol-water mixed solution contains 0.05V% formic acid.

12. The preparation method according to claim 1, characterized in that, In step S5, fraction B2 is separated by gel column chromatography to obtain fraction K3; based on a total volume of 100, the volume ratio of methanol to water in the methanol-aqueous solution when eluting fraction K3 is (65:35) - (75:25).

13. The preparation method according to claim 12, characterized in that, The volume ratio of methanol to water in the methanol-aqueous solution when eluting fraction K3 is 70:

30.

14. The preparation method according to claim 1, characterized in that, In step S6, the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is (44:56)-(48:52); and, the acetonitrile-water mixed solution contains 0.01-0.5 V% trifluoroacetic acid.

15. The preparation method according to claim 14, characterized in that, In step S6, the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is 46:

54.

16. The preparation method according to claim 14, characterized in that, In step S6, the acetonitrile-water mixed solution contains 0.1 V% trifluoroacetic acid.

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

18. Use of a pharmaceutical composition containing lappaconitine in the preparation of an antidepressant drug, characterized in that, Lappaconitine has the structure shown in formula (1): （1）。 19. The application according to claim 18, wherein The pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

20. The application according to claim 18, wherein The pharmaceutical composition further comprises a synergist; The synergist is one or several of the following substances: Fluoxetine, paroxetine, fluvoxamine, sertraline, citalopram, escitalopram, venlafaxine, duloxetine, mirtazapine, bupropion, agomelatine, trazodone, reboxetine, imipramine, amitriptyline, clomipramine, doxepin, maprotiline, moclobemide, Shugan Jieyu Capsule, Hypericum perforatum extract, Flupentixol Melitracen.

21. The application according to claim 18, wherein The dosage form of the pharmaceutical composition is tablets, capsules, granules, oral liquids, extracts, dripping pills or pellets.

22. Use of lappaconitine in the preparation of antidepressant drugs, characterized in that, Lappaconitine has the structure shown in formula (1): （1）。

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