CN118978484A - Lappaconitine, preparation method thereof, pharmaceutical composition and application thereof in antidepressant drugs - Google Patents

Abstract

The invention relates to the field of medical technology, and discloses lappaconitine and a preparation method thereof, a pharmaceutical composition and application thereof in antidepressant drugs; wherein the _C18 -type diterpene alkaloid lappaconitine provided by the invention exhibits significant antidepressant activity in a mouse behavioral despair model experiment, is significantly superior to a positive drug fluoxetine, and can be used for developing antidepressant drugs.

Description

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

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a C ₁₈ diterpene alkaloid compound lappaconitine, a preparation method thereof, a pharmaceutical composition and application thereof in antidepressant medicines.

Background

Nowadays, social competition is increasingly strong, high-intensity and high-pressure life has serious influence on physical and mental health of people, and depression is gradually increased and normal work and life of people are seriously influenced. At present, medicines for treating depression generally have larger side effects and have certain limitation in clinical application.

As a non-addictive analgesic drug which is first initiated in China, the lappaconitine has the advantages of good analgesic activity, long maintenance time and less adverse reaction, and is widely applied to the treatment of light and moderate pain such as postoperative pain, cancer pain and the like clinically. At present, the pharmacological activities of lappaconitine, such as antiarrhythmic, anti-inflammatory and anti-tumor activities, are also studied, but the antidepressant activity of lappaconitine has not been reported.

The inventor prepares lappaconitine (Lappaconitine) from a traditional Chinese medicine aconite, and finds that the lappaconitine shows remarkable antidepressant activity in a mouse behavior destimation model experiment, is superior to a positive medicine fluoxetine (Fluoxetine), and can be used for developing antidepressant medicines.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention provides a preparation method of C ₁₈ diterpene alkaloid lappaconitine, a pharmaceutical composition and application thereof in antidepressant drugs.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

Lappaconitine having a structure represented by formula (1):

The lappaconitine can be a compound comprising a structure shown in a formula (1), a stereoisomer, a tautomer or a pharmaceutically acceptable salt. Namely, compounds comprising the structure shown in the formula (1), stereoisomers, tautomers and pharmaceutically acceptable salts thereof are all within the scope of the application.

The invention also provides a preparation method of the lappaconitine, wherein the lappaconitine is obtained by extracting and separating from aconite.

In the above technical scheme, the preparation method of lappaconitine comprises the following steps:

S1, taking dried aconite, adding a solvent for reflux extraction, merging extracting solutions, and concentrating to obtain extractum;

s2, adding the extract into dilute hydrochloric acid for full dissolution, suspending, and filtering to remove impurities to obtain an acid aqueous solution; extracting the acid aqueous solution with dichloromethane for one time, and filtering the primary extract to obtain a filtered acid aqueous solution; adjusting the pH value of the filtered acid aqueous solution to be alkaline; then, performing secondary extraction by using dichloromethane to obtain a secondary extract;

S3, performing gradient elution on the secondary extract by using a dichloromethane-methanol solution to obtain a fraction B; when the silica gel thin layer is used for detection, the Rf value corresponding to the fraction B is 0.76-0.80;

s4, performing gradient elution on the fraction B by using a methanol-water mixed solution to obtain a fraction B2;

when the silica gel thin layer is used for detection, the Rf value corresponding to the fraction B2 is 0.45-0.49;

S5, carrying out gradient elution on the fraction B2 by using a methanol-water solution to obtain a fraction K3;

when the silica gel thin layer is used for detection, the K3 value corresponding to the fraction B2 is 0.64-0.76;

S6, separating the lappaconitine from the fraction K3 by using an RP-HPLC method (namely a reversed phase high performance liquid chromatography separation method); the mobile phase used in the RP-HPLC method is acetonitrile-water mixed solution.

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

In step S2, the extract is added into diluted hydrochloric acid with the mass of 8-15 times for full dissolution, suspension and filtration for impurity removal to obtain an acid aqueous solution.

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

Further, in step S3, the secondary extract is separated by silica gel column chromatography to obtain fraction B; eluting the dichloromethane-methanol solution (corresponding to fraction B) in the volume ratio of dichloromethane to methanol (97:3) - (93:7) based on the total volume of 100; preferably, the volume ratio of dichloromethane to methanol is 95:5.

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

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

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

Further, in step S5, the fraction B2 is separated by gel column chromatography to obtain a fraction K3; eluting the methanol-water solution (corresponding to the fraction K3) with the methanol-water solution with the total volume of 100 as a reference, wherein the volume ratio of the methanol to the water in the methanol-water solution is (65:35) - (75:25); preferably, the volume ratio of methanol to water is 70:30.

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

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 0.01-0.5V% of trifluoroacetic acid.

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

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

Further, in step S1, the solvent is 88-98V% ethanol water solution, the solvent is added with 8-10 times of the aconite root by mass, the times of reflux extraction are 2-4 times, and each time of extraction is 1-3 hours.

The invention also provides a pharmaceutical composition, which comprises the lappaconitine.

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

Further, the pharmaceutical composition further comprises a potentiating agent;

the synergist is one or more of the following substances:

Fluoxetine, paroxetine, fluvoxamine, sertraline, citalopram, escitalopram, venlafaxine, duloxetine, mirtazapine, bupropion, agomelatine, trazodone, reboxetine, imipramine, amitriptyline, chlorimipramine, doxepin, maprotiline, mollobemide, liver soothing capsules, san john's extract, flupentixol and the like.

That is, pharmaceutical compositions containing the lappaconitine of the present invention as an active ingredient and conventional pharmaceutical excipients or adjuvants or carriers are also included in the present invention.

Further, the dosage form of the pharmaceutical composition is tablets, capsules, granules, oral liquid, medicinal granules, dripping pills or micropills.

The invention also provides an application of the lappaconitine or the pharmaceutical composition in antidepressant drugs.

Compared with the prior art, the invention has the following advantages:

(1) Experimental results show that the lappaconitine provided by the invention has remarkable antidepressant activity in a mouse behavior despair model tail suspension experiment, is superior to a positive drug fluoxetine, and can be used for developing antidepressant drugs;

(2) The method for extracting lappaconitine from aconite with simple operation, good reproducibility and high extraction purity is provided.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent.

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

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

The terms "comprising," "including," or any other variation thereof, as used herein, 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 may include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.

The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase by regular vendors without the manufacturer's attention.

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.

Example 1

A preparation method of C ₁₈ diterpene alkaloid lappaconitine comprises the following steps:

S1, taking dried aconite, adding a solvent for reflux extraction, merging extracting solutions, and concentrating to obtain extractum; specifically, 200.5kg of dried aconite is taken, a 95% ethanol water solution with the mass being 10 times of that of the aconite is taken as a solvent for reflux extraction for three times, each time of extraction is 2 hours, and the extracts are combined and concentrated to obtain 6kg of extractum;

s2, adding the extract into dilute hydrochloric acid for full dissolution, suspending, and filtering to remove impurities to obtain an acid aqueous solution; extracting the acid aqueous solution with dichloromethane for one time, and filtering the primary extract to obtain a filtered acid aqueous solution; adjusting the pH value of the filtered acid aqueous solution to be alkaline; then, performing secondary extraction by using dichloromethane to obtain a secondary extract;

In the embodiment, the extract is added into 0.5% dilute hydrochloric acid (60L) with the mass of 10 times to be fully dissolved and suspended, and then the acid aqueous solution is obtained after filtration and impurity removal; the aqueous acid solution was extracted 3 times with 1.5 times the volume of dichloromethane to give 2.5kg of acidified dichloromethane primary extract (reject) and 1.2kg of filtered aqueous acid solution, i.e. aqueous acid layer, which was adjusted to ph=9 with sodium hydroxide, followed by extraction with dichloromethane again, this dichloromethane secondary extract 500g.

S3, performing gradient elution on the secondary extract by using a dichloromethane-methanol solution to obtain a fraction B; when the silica gel thin layer is used for detection, the Rf value corresponding to the fraction B is 0.76-0.80.

Through silica gel column chromatography, the volume ratio is 100: 0. 95: 5. 92: 8. 90: 10. 85: 15. 75: 25. 65: 35. 50: 50. 0: performing gradient elution on the secondary extract by using 100 methylene dichloride-methanol to sequentially obtain fractions 1-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80 and 81-90, collecting 90 fractions, then performing thin-layer detection on the obtained fractions by using silica gel, and combining similar fractions A, B, C, D, E, F, G, H and I sequentially according to the brick red spot observation Rf values of 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) shown by the bismuth iodide reagent; wherein, fractions 11-20 are combined to obtain fraction B.

Fraction B was selected for further isolation based on the brick red spot exhibited by the bismuth potassium iodide reagent and the characteristic ultraviolet absorption (λ _max =230 nm) of diterpene alkaloids observed by HPLC analysis.

S4, performing gradient elution on the fraction B by using a methanol-water mixed solution to obtain a fraction B2; when the silica gel thin layer is used for detection, the Rf value corresponding to the fraction B2 is 0.45-0.49. The volume ratio of methanol to water in the methanol-water mixed solution is (25:75) - (35:65) when fraction B2 is washed out; preferably, the volume ratio of methanol to water is 30:70. and the methanol-water mixed solution contains 0.01-1.1V% formic acid, preferably 0.05V% formic acid.

The specific operation comprises the following steps: fraction B was purified by ODS column chromatography using a volume ratio of 20: 80. 30: 70. 40: 60. 50: 50. 60: 40. 80: 20. 100:0 (containing 0.05% formic acid) to obtain fractions 1-6, 7-15, 16-23, 24-36, 37-45, 46-57, 58-68 in total, collecting 68 fractions, and combining similar fractions into 7 fractions B1-B7 according to the tile red spot observation Rf value of the bismuth potassium iodide reagent of 0.35-0.44 (fraction 1-6), 0.45-0.49 (fraction 7-15), 0.50-0.60 (fraction 16-23), 0.61-0.69 (fraction 24-36), 0.70-0.74 (fraction 37-45), 0.75-0.79 (fraction 46-57) and 0.80-0.89 (fraction 58-68) respectively. Fractions 7-15 were combined to give fraction B2.

Fraction B2 was selected for further isolation based on the brick red spot exhibited by the bismuth potassium iodide reagent and the characteristic ultraviolet absorption (λ _max =230 nm) of diterpene alkaloids observed by HPLC analysis.

S5, carrying out gradient elution on the fraction B2 by using a methanol-water solution to obtain a fraction K3; when the silica gel thin layer is used for detection, the K3 value corresponding to the fraction B2 is 0.64-0.76. Eluting fraction K3 (i.e. fraction K3 corresponds to) the methanol-water solution having a methanol to water volume ratio of (65:35) - (75:25); preferably, the volume ratio of methanol to water is 70:30.

The specific operation comprises the following steps: subjecting fraction B2 to Sephadex LH-20 gel column chromatography with a volume ratio of 0: 100. 30: 70. 70: 30. 100:0, sequentially collecting fractions 1-4, 5-13, 14-19 and 20-24, collecting 24 fractions, detecting by silica gel thin layer chromatography, respectively obtaining tile red spots according to bismuth potassium iodide reagent with Rf values of 0.30-0.55 (fraction 1-4), 0.56-0.63 (fraction 5-13), 0.64-0.76 (fraction 14-19) and 0.77-0.85 (fraction 20-24), and mixing similar fractions to obtain 4 fractions K1-K4; fractions 14-19 were combined to give fraction K3.

In this step, fraction K3 was selected for the next separation based on the brick red spot exhibited by the bismuth potassium iodide reagent and the characteristic ultraviolet absorption (λ _max =230 nm) of diterpene alkaloids observed by HPLC analysis.

S6, separating the lappaconitine from the fraction K3 by using an RP-HPLC method; the mobile phase used in the RP-HPLC method is 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% of trifluoroacetic acid. Preferably, the volume ratio of acetonitrile to water in the acetonitrile-water mixed solution is 46:54, the acetonitrile-water mixed solution contains 0.1V% trifluoroacetic acid.

The specific operation comprises the following steps: acetonitrile-water mixed solution (containing 0.1% of trifluoroacetic acid) is used as a mobile phase, and an HPLC method is adopted, wherein the volume ratio of acetonitrile to water is 46:54, lappaconitine (t _R =18.5 min, 95% purity) was prepared in fraction K3 using a C ₁₈ column, defined as compound 1.

Physical properties and detection data of the compound 1 produced in example 1 are as follows:

White amorphous powder, easily soluble in methanol. High resolution mass spectrum (HR-ESI-MS) M/z585.3160 ([ M+H ] ⁺ calculated as 585.3170), the molecular formula was determined to be C ₃₂H₄₄N₂O₈ in combination with nuclear magnetic data, the unsaturation was calculated as 12, and the nuclear magnetic data were as follows:

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

¹³C NMR(100MHz,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).

the structural formula of the C ₁₈ diterpene alkaloid lappaconitine is shown as the following formula:

Comparative example 1

Comparative example 1 of the present invention provides a process for preparing lappaconitine, a type C ₁₈ diterpene alkaloid, which is similar to example 1, except that in step S6, trifluoroacetic acid is not contained in an aqueous solution of a mobile phase used in separating fraction K3 by RP-HPLC.

The compound prepared in comparative example 1 has serious peak tailing in the preparation process, and the prepared compound 1 has purity less than 50% after liquid phase detection.

Comparative example 2

The present invention provides a method for preparing lappaconitine, a diterpene alkaloid of C ₁₈, in comparative example 2, which is similar to example 1, except that in step S4, when fraction B is subjected to ODS column chromatography, an initial volume ratio of 20:80 (no formic acid) in methanol-water. Comparative example 2 failed to detect this compound during the subsequent fraction preparation, and the results showed that compound 1 could not be prepared.

Test example 1

The test example discloses the antidepressant effect of the compound lappaconitine in a mouse behavior destimation model experiment (gold standard for antidepressant drug screening).

1. Experimental materials and instruments

Fluoxetine hydrochloride was purchased from Shanghai source leaf biotechnology limited; the norlinderane and the methemodin are purchased from Shanghai Shidand standard technical service Co., ltd; radix Aconiti lateralis total alkaloids; lappaconitine.

ZIL-2 mice were independently movable box (Shanghai Xinman science education equipments Co., ltd.);

YLS-18A mouse tail suspension instrument (Anhua Zhenghua biological instrument Co., ltd.); electronic balance (Beijing Sidoriko instruments Co.).

2. Experimental animal

ICR mice, male, body weight 18-22g, spf grade, supplied by velocin, animal license number: SYXK (Beijing) 2023-0001 animals are kept in an environment with the temperature of 23+/-2 ℃ and the humidity of 50% +/-10%, and the illumination time is 12 hours per day (7:00-19:00 illumination). After 3d of animal feeding, each experiment was performed, fasted 6 hours before the start of the behavioural experiment, and free water was consumed.

3. Experimental method

70 ICR male mice were divided into 7 groups, namely a blank group (physiological saline with the same volume), a fluoxetine hydrochloride group (10 mg/kg), a methemodin group (10 mg/kg), a norlinderamine group (10 mg/kg), a aconite total alkaloid group (10 mg/kg), a lappaconitine low dose group (0.03 mg/kg) and a lappaconitine high dose group (0.3 mg/kg) in descending order of animal screening autonomous activity times; each group of 10 is numbered separately. The drugs are added with normal saline to prepare suspension, each group is administrated by stomach irrigation once a day, the administration volume is 0.1ml/10g body weight, and after continuous administration for 3 days, autonomous activity experiments are carried out on the 4 th day and tail suspension experiments are carried out on the 5 th day.

3. Experimental results

As shown in Table 1, compared with the blank group, the low-dose and high-dose group (0.03 and 0.3 mg/kg) of lappaconitine can obviously shorten the immobility time of mice in the tail suspension experiment, and shows obvious antidepressant activity, which is obviously superior to the positive drugs fluoxetine (10 mg/kg, the first-line chemical for clinically treating depression), methyloxflat base (10 mg/kg), norlinderane (10 mg/kg) and aconite total alkaloids (10 mg/kg).

TABLE 1 Effect of lappaconitine on mice tail-holding time in behavioural despair model

(Mean.+ -. Standard deviation; ^*** P <0.001 compared to blank)

As shown in table 2, each of the dosing groups had no significant effect on the voluntary activity of mice in the open field experiment compared to the blank group, indicating that the effect of each dosing group on the immobility time of mice in the tail suspension experiment was independent of the neuroexcitability.

TABLE 2 influence of lappaconitine on the horizontal movement distance of mice in the behavior destimation model

In conclusion, the C ₁₈ diterpene alkaloid lappaconitine has anti-depression activity obviously superior to that of first-line medicine fluoxetine for depression, and can be used as a prodrug for treating depression.

Application example 1

The application example of the invention discloses a capsule taking lappaconitine as a raw material medicine, which comprises the following components:

the preparation process comprises the following steps:

Mixing lappaconitine, starch and sodium metabisulfite, adding absolute ethanol, making into soft mass, sieving with 24 mesh sieve, granulating, drying, adding magnesium stearate, mixing, and making into capsule.

Application example 2

The application example of the invention discloses a granule taking a compound lappaconitine as a raw material medicine, which comprises the following components:

Lappaconitine 4.0mg

Starch 6.0g

Sodium bisulphite 0.2g

Magnesium stearate 0.2g

Proper amount of absolute ethyl alcohol

Making into 100 bags.

The preparation process comprises the following steps:

Mixing lappaconitine with starch and sodium bisulphite, adding absolute ethanol to obtain soft mass, sieving with 24 mesh sieve, granulating, drying, adding magnesium stearate, mixing, and bagging.

Application example 3

The application example of the invention discloses an oral liquid taking a compound lappaconitine as a raw material medicine, which comprises the following components:

the preparation process comprises the following steps:

mixing the above components, preparing into oral liquid by conventional method, and packaging.

Application example 4

The application example of the invention discloses an injection taking compound lappaconitine as raw material medicines, which comprises the following components:

the preparation process comprises the following steps:

After the components are evenly mixed, 100 pieces can be obtained by adopting the conventional preparation method of injection.

Application example 5

The application example of the invention discloses a tablet taking compound lappaconitine and fluoxetine as raw material medicines, which comprises the following components:

the preparation process comprises the following steps:

Mixing lappaconitine, fluoxetine, hydroxypropyl methylcellulose, pulvis Talci, lactose, and magnesium stearate, adding absolute ethanol, making into soft mass, sieving with 24 mesh sieve, granulating, drying, adding magnesium stearate, mixing, and tabletting.

Application example 6

The application example of the invention discloses a capsule taking compound lappaconitine and lulutide as raw material medicines, which comprises the following components:

the preparation process comprises the following steps:

Mixing lappaconitine, luyou Thai and sodium metabisulfite, adding anhydrous ethanol, making into soft mass, sieving with 24 mesh sieve, granulating, drying, adding magnesium stearate, mixing, and making into capsule.

Application example 7

The application example of the invention discloses an injection taking compounds of lappaconitine and paroxetine as bulk drugs, which comprises the following components:

the preparation process comprises the following steps:

After the components are evenly mixed, 100 pieces can be obtained by adopting the conventional preparation method of injection.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention.

It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (10)

1. Lappaconitine is characterized by having a structure represented by formula (1):

2. A method for preparing lappaconitine according to claim 1, wherein lappaconitine is obtained by extraction and separation from aconite.

3. The preparation method according to claim 2, characterized by comprising the steps of:

S1, taking dried aconite, adding a solvent for reflux extraction, merging extracting solutions, and concentrating to obtain extractum;

s2, adding the extract into dilute hydrochloric acid for full dissolution, suspending, and filtering to remove impurities to obtain an acid aqueous solution; extracting the acid aqueous solution with dichloromethane for one time, and filtering the primary extract to obtain a filtered acid aqueous solution; adjusting the pH value of the filtered acid aqueous solution to be alkaline; then, performing secondary extraction by using dichloromethane to obtain a secondary extract;

S3, performing gradient elution on the secondary extract by using a dichloromethane-methanol solution to obtain a fraction B; when the silica gel thin layer is used for detection, the Rf value corresponding to the fraction B is 0.76-0.80;

s4, performing gradient elution on the fraction B by using a methanol-water mixed solution to obtain a fraction B2; when the silica gel thin layer is used for detection, the Rf value corresponding to the fraction B2 is 0.45-0.49;

S5, carrying out gradient elution on the fraction B2 by using a methanol-water solution to obtain a fraction K3;

when the silica gel thin layer is used for detection, the K3 value corresponding to the fraction B2 is 0.64-0.76;

s6, separating the lappaconitine from the fraction K3 by using an RP-HPLC method;

the mobile phase used in the RP-HPLC method is acetonitrile-water mixed solution.

4. A method according to claim 3, wherein in step S2, the aqueous acid solution after filtration is adjusted to a pH of 8-11 with sodium hydroxide.

5. A method according to claim 3, wherein in step S2, the aqueous acid solution after filtration is adjusted to pH 9 with sodium hydroxide.

6. The method according to claim 3, wherein in step S2, the extract is added into 8-15 times of diluted hydrochloric acid for full dissolution, suspension and filtration to remove impurities to obtain an acid aqueous solution.

7. A process according to claim 3, wherein the dilute hydrochloric acid has a concentration of 0.1-1.0%.

8. The process according to claim 3, wherein in step S3, the secondary extract is separated by silica gel column chromatography to obtain fraction B; the volume ratio of methylene chloride to methanol in the methylene chloride-methanol solution in fraction B was eluted at (97:3) - (93:7) based on a total volume of 100.

9. A pharmaceutical composition, characterized in that, comprising lappaconitine according to claim 1.

10. Use of lappaconitine according to claim 1 and/or a pharmaceutical composition according to claim 9 in antidepressants.