Diterpenoid alkaloid bulleyaconitine A and preparation method, pharmaceutical composition and application thereof

Abstract

The application relates to the technical field of medicines, and discloses a diterpene alkaloid lappaconitine, a preparation method, a pharmaceutical composition and application. The diterpene alkaloid lappaconitine provided by the application shows significant antidepressant activity in a mouse behavioral despair model tail suspension test, is obviously superior to a positive drug fluoxetine, and can be used for developing antidepressant drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and more particularly relates to a C 19 type diterpenoid alkaloid bulleyaconitine A, a preparation method, a pharmaceutical composition and application in antidepressant drugs. BACKGROUND

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

[0003] The inventors prepared bulleyaconitine A from a traditional Chinese medicine aconite, found that it showed significant antidepressant activity in a mouse behavioral despair model experiment, and was significantly better than the positive drug fluoxetine, and can be used for developing antidepressant drugs. SUMMARY

[0004] In view of the deficiencies in the prior art, the present application provides a diterpenoid alkaloid bulleyaconitine A (specifically, a C 19 type diterpenoid alkaloid bulleyaconitine A), a preparation method, a pharmaceutical composition and application in antidepressant drugs.

[0005] To achieve the above-mentioned purpose, the technical scheme of the present application is as follows:

[0006] A diterpenoid alkaloid bulleyaconitine A has the following structure shown in formula (1):

[0007]

[0008] It should be noted that the diterpenoid alkaloid bulleyaconitine A can be a compound including the structure shown in formula (1), a stereoisomer, a tautomer or a pharmaceutically acceptable salt. That is, the compound including the structure shown in formula (1), its stereoisomer, tautomer and pharmaceutically acceptable salt are all within the protection scope of the present application.

[0009] The present application also provides a preparation method of the diterpenoid alkaloid bulleyaconitine A, which is obtained by extraction and separation from aconite.

[0010] In the above technical scheme, the preparation method of the diterpenoid alkaloid bulleyaconitine A comprises the following steps:

[0011] S1, taking dried aconite, adding a solvent to reflux extraction, combining the extraction liquid and concentrating to obtain an extract;

[0012] S2, the extract is added to dilute hydrochloric acid to dissolve and suspend, then filtered to remove impurities to obtain an acid aqueous solution;

[0013] The acid aqueous solution is extracted once with dichloromethane, and after filtering out the first extract, a filtered acid aqueous solution is obtained.

[0014] The pH of the filtered acid aqueous solution is adjusted to be alkaline, and then extracted twice with dichloromethane to obtain a second extract.

[0015] S3, the second extract is gradient eluted with dichloromethane-methanol solution to obtain fraction B.

[0016] When using silica gel thin layer chromatography, the Rf value corresponding to fraction B is 0.76-0.80.

[0017] S4, fraction B is gradient eluted with methanol-water mixed solution to obtain fraction B4.

[0018] When using silica gel thin layer chromatography, the Rf value corresponding to fraction B4 is 0.61-0.69.

[0019] S5, fraction B4 is gradient eluted with methanol-water solution to obtain fraction L2.

[0020] When using silica gel thin layer chromatography, the Rf value corresponding to fraction L2 is 0.53-0.65.

[0021] S6, the diterpene alkaloid aconitine is separated from fraction L2 by RP-HPLC method (i.e. reverse phase high performance liquid chromatography method).

[0022] The mobile phase used in the RP-HPLC method is a mixture of acetonitrile and water.

[0023] Further, in step S2, the filtered acid aqueous solution is adjusted to pH 8-11 with sodium hydroxide; preferably, the pH is adjusted to 9.

[0024] Further, in step S2, the extract is added to 8-15 times the mass of dilute hydrochloric acid to dissolve and suspend, then filtered to remove impurities to obtain an acid aqueous solution.

[0025] Further, the concentration of the dilute hydrochloric acid is 0.1-1.0%; preferably 0.5%.

[0026] 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, the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solution used to elute fraction B (i.e. corresponding to fraction B) is (97:3)-(93:7); preferably, the volume ratio of dichloromethane to methanol is 95:5.

[0027] Further, in step S4, the fraction B is separated by ODS column chromatography to obtain fraction B4; the volume ratio of methanol to water in the methanol-water mixed solution used to elute fraction B4 (i.e. corresponding to fraction B4) is (45:55)-(55:45); preferably, the volume ratio of methanol to water is 50:50.

[0028] Further, in step S4, the methanol-water mixed solution contains 0.01-1.1V% formic acid.

[0029] Preferably, in step S4, the methanol-water mixed solution contains 0.05V% formic acid.

[0030] Further, in step S5, the fraction B4 is separated by gel column chromatography to obtain fraction L2; the volume ratio of methanol to water in the methanol-water solution used to elute fraction L2 (i.e. corresponding to fraction L2) is (25:75)-(35:65) based on 100 total volume; preferably, the volume ratio of methanol to water is 30:70.

[0031] Further, in step S6, the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is (40:60)-(45:55); and the acetonitrile-water mixed solution contains 0.01-0.5V% trifluoroacetic acid.

[0032] Preferably, in step S6, the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is 43:57.

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

[0034] Further, in step S1, the solvent is 88-98V% aqueous ethanol solution, the solvent is added in 8-10 times of the mass of the subject, and the reflux extraction is performed 2-4 times, each time for 1-3h.

[0035] In another aspect, the present application also provides a pharmaceutical composition comprising the above-mentioned diterpene alkaloid, grass mallow root.

[0036] Further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0037] Further, the pharmaceutical composition further comprises a synergist, which is one or more of the following: fluoxetine, paroxetine, fluvoxamine, sertraline, citalopram, escitalopram, venlafaxine, duloxetine, mirtazapine, bupropion, agomelatine, trazodone, reboxetine, imipramine, clomipramine, desipramine, maprotiline, moclobemide, Shugan Jieyu Capsule, St. John's wort extract, flupentixol melitracen, etc.

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

[0039] Further, the dosage form of the pharmaceutical composition is a tablet, a capsule, a granule, an oral solution, a powder, a dripping pill or a pellet.

[0040] In another aspect of the present application, the above-mentioned C 19 application of the C

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

[0042] (1) The experimental results show that the C 19 type diterpene alkaloid aconitum ferox provided by the present application shows 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 antidepressants;

[0043] (2) The present application provides a preparation method of the C 19 type diterpene alkaloid aconitum ferox, which can be obtained from aconite with simple operation, good reproducibility, high extraction purity and the like. 19 type diterpene alkaloid aconitum ferox. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be further described in detail below in combination with embodiments.

[0045] It should be understood that the specific embodiments described herein are merely intended to explain the present application, and are not intended to limit the present application.

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

[0047] As used herein, the terms "comprise", "comprising", or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements, but can include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.

[0048] In the embodiments, unless otherwise specified, the techniques or conditions are performed according to the techniques or conditions described in the literature in the art, or according to the product instructions. Unless otherwise specified, the reagents or instruments used are conventional products that can be purchased through regular channels.

[0049] The endpoints of the ranges and any values disclosed herein are not limited to the precise values recited as the exact dimensions are not critical to the invention. Any numeric range recited is intended to include all values from the lower value to the upper value, inclusive of both values, and to sub-ranges falling within the specified range. In this context, individual points within a range should not be dismissed from the scope of the range. For numeric ranges, the endpoints of each range, the endpoints of each range and individual point values, and individual point values can be combined with each other to yield one or more new numeric ranges, which should be considered as specifically disclosed herein.

[0050] Furthermore, the technical features involved in each of the embodiments of the application described below can be combined with each other as long as there is no conflict.

[0051] Embodiment 1

[0052] The present embodiment discloses a preparation method of C 19 type diterpenoid alkaloid lappaconitine, the C 19 type diterpenoid alkaloid lappaconitine is obtained by extraction and separation from aconite.

[0053] The preparation method of the C 19 type diterpenoid alkaloid lappaconitine comprises the following steps:

[0054] S1, taking dry aconite, adding solvent to reflux extraction, and then combining the extraction liquid and concentrating to obtain an extract;

[0055] Specifically, taking dry aconite, and taking 95% ethanol aqueous solution with a mass of 10 times of aconite as a solvent, reflux extraction for three times, each time for 2h, combining the extraction liquid and concentrating to obtain an extract;

[0056] S2, after the extract is added to dilute hydrochloric acid and fully dissolved and suspended, impurities are removed by filtration to obtain an acid aqueous solution; the acid aqueous solution is extracted once with dichloromethane, and after the first extraction is filtered out, a filtered acid aqueous solution is obtained; the pH of the filtered acid aqueous solution is adjusted to be alkaline; and then the filtered acid aqueous solution is twice extracted with dichloromethane to obtain a second extraction.

[0057] Specifically, after the extract is added to 10 times of 0.5% dilute hydrochloric acid and fully dissolved and suspended, impurities are removed by filtration, and the filtered acid aqueous solution is extracted with 1.5 times of dichloromethane three times to obtain acid dichloromethane extract (first extraction, discarded) and an acid water layer, which is the filtered acid aqueous solution;

[0058] The filtered acid aqueous solution is adjusted to pH 8-11 with sodium hydroxide; preferably, the pH is adjusted to 9; and then the filtered acid aqueous solution is twice extracted with dichloromethane to obtain a second dichloromethane extraction.

[0059] S3, gradient elution of the secondary extract with dichloromethane-methanol solution to obtain fraction B; when using silica gel thin layer chromatography, the Rf value of the brick red spot shown by the bismuth potassium iodide reagent corresponding to fraction B is observed to be 0.76-0.80. Specifically, 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).

[0060] In this embodiment, preferably, the secondary extract is separated by silica gel column chromatography using 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, respectively.

[0061] The Rf values of the brick red spots shown by the bismuth potassium iodide reagent are 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), and 0.25-0.28 (fractions 81-90) when using silica gel thin layer chromatography, and similar fractions are combined to obtain fractions A, B, C, D, E, F, G, H, and I, respectively. The fractions 11-20 are combined to obtain fraction B.

[0062] S4, gradient elution of fraction B with methanol-water mixed solution to obtain fraction B4; the methanol-water mixed solution contains 0.05V% formic acid. The Rf value of fraction B4 is 0.61-0.69 when using silica gel thin layer chromatography.

[0063] That is, fraction B is selected for further separation based on the brick red spot shown by the bismuth potassium iodide reagent and the characteristic ultraviolet absorption (λ max = 230 nm) of diterpene alkaloids observed by HPLC analysis.

[0064] Specifically, fraction B is separated by ODS column chromatography to obtain fraction B4; the volume ratio of methanol to water in the methanol-water mixed solution when eluting fraction B4 (i.e., corresponding to fraction B4) is (45:55)-(55:45). The methanol-water mixed solution contains 0.01-1.1V% formic acid.

[0065] In this embodiment, preferably, a methanol-water mixed solution (containing 0.05% formic acid in the methanol-water mixed solution) with a volume ratio of 20:80, 30:70, 40:60, 50:50, 60:40, 80:20, 100:0 is used for gradient elution. Then, fractions 1-6, 7-15, 16-23, 24-36, 37-45, 46-57, 58-68 are obtained in sequence, and 68 fractions are collected in total.

[0066] Using silica gel thin layer chromatography, 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) according to the brick red spots shown by the bismuth potassium iodide reagent, and similar fractions are combined into 7 fractions B1-B7. Among them, fractions 24-36 are combined to obtain fraction B4. The Rf value of fraction B4 is 0.61-0.69.

[0067] S5, fraction B4 is subjected to gradient elution using a methanol-water solution to obtain fraction L2; when using silica gel thin layer chromatography, the Rf value corresponding to fraction L2 is 0.53-0.65;

[0068] According to the brick red spots shown by the bismuth potassium iodide reagent and the characteristic ultraviolet absorption (λ max = 230 nm) of diterpene alkaloids observed by HPLC analysis, fraction B4 is selected for further separation. Among them, fraction B4 is subjected to gel column chromatography to obtain fraction L2; based on a total volume of 100, the volume ratio of methanol to water in the methanol-water solution when eluting fraction L2 is (25:75)-(35:65).

[0069] The specific operation of this embodiment includes: fraction B4 is subjected to gel column chromatography, gradient elution is performed using a methanol-water solution with a volume ratio of 0:100, 30:70, 70:30, 100:0, fractions 1-6, 7-15, 16-21, 22-25 are collected in sequence, and 25 fractions are collected in total. Then, using silica gel thin layer chromatography, the Rf values are observed to be 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) according to the brick red spots shown by the bismuth potassium iodide reagent, and similar fractions are combined into 4 fractions L1-L4; fraction 7-15 is combined to obtain fraction L2.

[0070] S6, C 19The compound 1 is a type of diterpene alkaloid. The mobile phase used in the RP-HPLC method is a mixed solution of acetonitrile and water. The volume ratio of acetonitrile to water in the mixed solution of acetonitrile and water is (40:60)-(45:55). The mixed solution of acetonitrile and water contains 0.01-0.5V% trifluoroacetic acid.

[0071] Specifically, fraction L2 was selected for the next step of separation according to the brick red spot shown by the potassium bismuth iodide reagent and the characteristic ultraviolet absorption (λ max 230nm) of diterpene alkaloids observed by HPLC analysis. The volume ratio of acetonitrile to water in the mobile phase used in the RP-HPLC method for separating fraction L2 is 43:57. The mixed solution of acetonitrile and water contains 0.1V% trifluoroacetic acid. The C 18 chromatographic column was prepared from fraction L2. The type of diterpene alkaloid obtained from fraction L2 is t 19 R =22.5min, purity 98%).

[0072] The physical properties and detection data of the compound 1 prepared in Example 1 are as follows:

[0073] White amorphous powder, easily soluble in methanol. High resolution mass spectrometry (HR-ESI-MS) m / z 644.3396 ([M+H] + The calculated value is 644.3429, combined with nuclear magnetic data, to determine the molecular formula of C 35 H 49 NO 10 The unsaturation degree is calculated to be 12, and the nuclear magnetic data is as follows:

[0074] 1 H NMR (400MHz, CDCl3) δ: 8.01 (2H, d, J = 8.4Hz, H-2”, 6”), 6.92 (2H, d, J = 8.4Hz, H-3”, 5”), 4.87 (1H, d, J = 5.0Hz, H-14β), 3.97 (1H, d, J = 6.8Hz, H-6β), 3.85 (3H, s, 4”-OCH3), 3.59 (1H, d, J = 8.4Hz, H-18), 3.52, 3.27, 3.25, 3.14 (each 3H, s, 4×OCH3), 3.38 (1H, dd, J = 8.8, 6.0Hz, H-16), 1.33 (3H, s, H-2’), 1.09 (3H, t, J = 7.2Hz, H-21);

[0075] 13 ​C NMR (100 MHz, CDC13) δ: 169.9 (C-1'), 163.3 (C-4"), 166.1 (C-7"), 131.7 (C-2", 6"), 122.7 (C-1"), 113.7 (C-3", 5"), 85.7 (C-8), 85.1 (C-1), 83.6 (C-16), 83.0 (C-6), 78.6 (C-14), 78.5 (C-18), 74.9 (C-13), 62.0 (C-17), 59.1 (18-OCH3), 58.8 (16-OCH3), 57.8 (6-OCH3), 56.3 (1-OCH3), 55.4 (4"-OCH3), 53.6 (C-19), 50.3 (C-11), 49.5 (C-5), 49.1 (C-7), 49.1 (C-20), 45.2 (C-9), 41.0 (C-10), 39.3 (C-15), 39.2 (C-4), 35.8 (C-3), 34.9 (C-12), 26.4 (C-2), 21.7 (C-2'), 13.6 (C-21).

[0076] The C 19 The structural formula of the C

[0077]

[0078] Comparative Example 1

[0079] Comparative Example 1 of the present application provides a preparation method of the C 19 The preparation method of the C

[0080] The compound prepared in Comparative Example 1 has a serious peak tailing in the preparation process, and the analysis of liquid phase after preparation shows that there is a large amount of impurities, and the purity of the prepared compound 1 is less than 50%.

[0081] Comparative Example 2

[0082] Comparative Example 2 of the present application provides a preparation method of the C 19 The preparation method of the C

[0083] The compound prepared in Comparative Example 2 cannot be detected in the subsequent fraction preparation process:

[0084] The results show that the compound 1 cannot be prepared.

[0085] Test Example 1

[0086] This test example discloses the antidepressant effect of the above-mentioned compound, aconitine, in a mouse behavioral despair model experiment (the gold standard for screening antidepressants).

[0087] 1. Experimental materials and instruments

[0088] Fluoxetine hydrochloride was purchased from Shanghai Yuan Ye Biotechnology Co., Ltd.; methyl norlaudanosine and norlaudanosine were purchased from Shanghai Shidan Standard Technology Service Co., Ltd.; total alkaloids of Fuzi; aconitine.

[0089] ZIL-2 type mouse self-activity box (Shanghai Xinman Teaching Equipment Co., Ltd.); YLS-18A type mouse tail suspension instrument (Anhui Zhenghua Biological Instrument Equipment Co., Ltd.); electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.).

[0090] 2. Experimental animals

[0091] ICR mice, male, body weight 18-22 g, SPF level, provided by Weitong Lihua, animal license number: SYXK(Jing)2023-0001 The animals were raised in an environment with a temperature of 23±2℃ and a humidity of 50±10%, and the lighting time was 12h per day (lighting from 7:00 to 19:00). The animals were raised for 3 days before the experiments, and the behavior experiments started 6h after fasting, with free access to water.

[0092] 3. Experimental method

[0093] 70 ICR male mice were arranged in a snake shape in descending order of the number of self-activity times, and divided into 7 groups, namely the blank group (same volume of normal saline), the positive drug fluoxetine group (10 mg / kg), the methyl norlaudanosine group (10 mg / kg), the norlaudanosine group (10 mg / kg), the total alkaloids of Fuzi group (10 mg / kg), the low-dose aconitine group (0.03 mg / kg), and the high-dose aconitine group (0.3 mg / kg), 10 mice in each group, numbered respectively. The drugs were added to normal saline to prepare a solution, and each group was given intragastric administration once a day, with a drug volume of 0.1 ml / 10 g of body weight. After 3 days of continuous administration, the self-activity experiment was performed on the 4th day, and the tail suspension experiment was performed on the 5th day.

[0094] 3. Experimental results

[0095] As shown in Table 1, compared with the blank group, the low and high dose groups (0.03, 0.3 mg / kg) of the aconitum alkaloid can significantly shorten the immobile time of the mice in the tail suspension test, and show significant antidepressant activity, which is obviously better than the positive drug fluoxetine (10 mg / kg, a first-line chemical drug for clinical treatment of depression), methylcytisine (10 mg / kg), norlaudanosine (10 mg / kg) and fuzi total alkaloids (10 mg / kg).

[0096] Table 1 Influence of aconitum alkaloid on the immobile time of the tail suspension test of the behavior despair model mice

[0097]

[0098] (Compared with the blank group, *** P<0.001)

[0099] As shown in Table 2, compared with the blank group, the self-activity of the mice in the open field test in each administration group has no significant influence, which indicates that the effect of the immobile time of the mice in the tail suspension test in each administration group is irrelevant to the nerve excitability.

[0100] Table 2 Influence of aconitum alkaloid on the horizontal movement distance of the open field of the behavior despair model mice

[0101]

[0102]

[0103] In summary, the aconitum alkaloid C 19 The diterpene alkaloid aconitum alkaloid has significant antidepressant activity better than the first-line drug fluoxetine for depression, and can be applied as a drug precursor for treating depression.

[0104] Application Example 1

[0105] The application example discloses a capsule of aconitum alkaloid as a raw drug, and components thereof are as follows:

[0106]

[0107] The specific preparation process is as follows:

[0108] The aconitum alkaloid, starch and sodium pyrosulfite are uniformly mixed, anhydrous ethanol is added to prepare a soft material, the soft material is passed through a 24-mesh sieve to prepare granules, the granules are dried, magnesium stearate is added, and the mixture is uniformly mixed and loaded into a capsule.

[0109] Application Example 2

[0110] The application example discloses a granule of aconitum alkaloid as a raw drug, and components thereof are as follows:

[0111] Aconitum alkaloid 4.0 mg

[0112] Starch 6.0 g

[0113] Sodium bisulfite 0.2 g

[0114] Magnesium stearate 0.2 g

[0115] Anhydrous ethanol q.s.

[0116] Make 100 bags.

[0117] The specific preparation process is as follows:

[0118] Take aconitine and starch, sodium bisulfite, mix evenly, add anhydrous ethanol to make soft material, pass through 24 mesh sieve, make granules, dry, add magnesium stearate, mix evenly, and pack.

[0119] Application Example 3

[0120] The application example of the application discloses an oral liquid taking compound aconitine as raw material, and its components are as follows:

[0121]

[0122] The specific preparation process is as follows:

[0123] After mixing the above components, the oral liquid is prepared by the conventional preparation method, and is then divided and packaged.

[0124] Application Example 4

[0125] The application example of the application discloses an injection taking compound aconitine as raw material, and its components are as follows:

[0126]

[0127] The specific preparation process is as follows:

[0128] After mixing the above components, the injection is prepared by the conventional preparation method, and then 100 injections are obtained.

[0129] Application Example 5

[0130] The application example of the application discloses a tablet taking compound aconitine and fluoxetine as raw materials, and its components are as follows:

[0131]

[0132] The specific preparation process is as follows:

[0133] Take aconitine, fluoxetine, and hydroxypropyl methyl cellulose, talc, lactose, and magnesium stearate, mix evenly, add anhydrous ethanol to make soft material, pass through 24 mesh sieve, make granules, dry, add magnesium stearate, mix evenly, and press into tablets.

[0134] Application Example 6

[0135] The application example discloses a capsule taking compound aconitine and Lu You Tai as raw medicine, and components thereof are as follows:

[0136]

[0137]

[0138] The specific preparation process is as follows:

[0139] After mixing aconitine, Lu You Tai and sodium pyrosulfite, anhydrous ethanol is added to prepare soft material, which is sieved through a 24-mesh sieve, dried, and then magnesium stearate is added and mixed to be filled into capsules.

[0140] Application Example 7

[0141] The application example discloses an injection taking compound aconitine and paroxetine as raw medicine, and components thereof are as follows:

[0142]

[0143] The specific preparation process is as follows:

[0144] After mixing the above components, 100 injections can be obtained by using the conventional preparation method of injection.

[0145] The above examples only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as the limitation of the scope of the patent.

[0146] It should be noted that for those skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A method for preparing diterpenoid alkaloid chansuine A, characterized in that, The diterpene alkaloid aconitine has a structure as shown in formula (1): The diterpene alkaloid aconitine is obtained by extraction and separation from aconite; The preparation method comprises the following steps: S1, taking dried aconite, adding solvent to reflux extraction, and then combining and concentrating the extraction liquid to obtain an extract; S2, adding the extract to dilute hydrochloric acid to fully dissolve and suspend, filtering impurities to obtain an acid aqueous solution; The acid aqueous solution is extracted once with dichloromethane, and after filtering out the first extraction, a filtered acid aqueous solution is obtained; The pH of the filtered acid aqueous solution is adjusted to be alkaline, and then the solution is extracted twice with dichloromethane to obtain a second extraction; S3, gradient elution of the second extraction with dichloromethane-methanol solution to obtain fraction B; When identified by silica gel thin layer chromatography, the Rf value corresponding to fraction B is 0.76-0.80; The volume ratio of dichloromethane to methanol in the dichloromethane-methanol solution used to elute fraction B is (97:3)-(93:7) based on a total volume of 100. S4, gradient elution of fraction B with methanol-water mixed solution to obtain fraction B4; When identified by silica gel thin layer chromatography, the Rf value corresponding to fraction B4 is 0.61-0.69; The fraction B is separated by ODS column chromatography to obtain fraction B4; the volume ratio of methanol to water in the methanol-water mixed solution used to elute fraction B4 is (45:55)-(55:45). The methanol-water mixed solution contains 0.01-1.1V% formic acid. S5, gradient elution of fraction B4 with methanol-water solution to obtain fraction L2; When identified by silica gel thin layer chromatography, the Rf value corresponding to fraction L2 is 0.53-0.65; The fraction B4 is separated by gel column chromatography to obtain fraction L2; the volume ratio of methanol to water in the methanol-water solution used to elute fraction L2 is (25:75)-(35:65) based on a total volume of 100. S6, using C 18 The diterpene alkaloid lappaconitine was isolated from the fraction L2 by chromatography on a column 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 acetonitrile-water mixed solution is (40:60)-(45:55); and the acetonitrile-water mixed solution contains 0.01-0.5V% trifluoroacetic acid.

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

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

4. The method of claim 1, wherein, In step S2, the extract is added to 8-15 times the mass of dilute hydrochloric acid to fully dissolve and suspend, and then impurities are filtered out to obtain an acid 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 method of claim 1, wherein, The concentration of the dilute hydrochloric acid is 0.5%.

7. 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 used to elute fraction B is 95:

5.

8. The method of claim 1, wherein, In step S4, the volume ratio of methanol to water in the methanol-water mixed solution used to elute fraction B4 is 50:

50.

9. The method of claim 1, wherein, In step S4, the methanol-water mixed solution contains 0.05V% formic acid.

10. The method of claim 1, wherein, In step S5, the volume ratio of methanol to water in the methanol-water solution used to elute fraction L2 is 30:

70.

11. The method of claim 1, wherein, In step S6, the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is 43:

57.

12. The method of claim 1, wherein, In step S6, the acetonitrile-water mixed solution contains 0.1V% trifluoroacetic acid.

13. The method of claim 1, wherein, In step S1, the solvent is 88-98V% ethanol aqueous solution, the solvent is added with 8-10 times of the mass of the adduct, and the reflux extraction is performed 2-4 times, each time for 1-3 hours.

14. Use of diterpenoid alkaloid lappaconitine or a pharmaceutical composition comprising diterpenoid alkaloid lappaconitine in the preparation of an antidepressant medicament, characterized in that, The diterpene alkaloid lappaconitine has a structure as shown in formula (1):

15. Use according to claim 14, characterized in that, The pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

16. The use according to claim 14, characterized in that, The dosage form of the pharmaceutical composition is tablet, capsule, granule, oral liquid, powder, dripping pill or micro-pill.

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