New skeleton diterpene alkaloid compounds and preparation method and application thereof

Abstract

The present invention discloses a novel skeleton diterpene alkaloid compound and a preparation method and application thereof, wherein the novel skeleton cleaved ring C 20 The diterpene alkaloid compound Carmiseconapline A showed significant antidepressant activity in the mouse behavioral despair model experiment, which was significantly better than the positive drug fluoxetine and can be used to develop antidepressant drugs.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a new skeleton diterpene alkaloid compound and a preparation method and application thereof. Background Art

[0002] Nowadays, social competition is becoming increasingly fierce. 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, drugs for treating depression generally have large side effects and have certain limitations in clinical application.

[0003] The inventors prepared a novel carbon skeleton-split ring C from the traditional Chinese medicine Aconite 20 The diterpene alkaloid compound Carmiseconapline A was found to have significant antidepressant activity in a mouse behavioral despair model experiment, which was superior to the positive drug fluoxetine and can be used to develop antidepressant drugs. Summary of the invention

[0004] The purpose of the present invention is to overcome the defects in the prior art and provide a split ring C having a new skeleton. 20 A diterpene alkaloid compound Carmiseconapline A, a preparation method thereof, a pharmaceutical composition and application thereof.

[0005] To achieve the above-mentioned purpose, the technical solution of the present invention provides a new skeleton diterpene alkaloid compound (referred to as compound Carmiseconapline A), having a structure as shown in formula (1):

[0006]

[0007] The split ring C 20 Diterpene alkaloid compounds are extracted and separated from Radix Aconiti Lateralis Preparata.

[0008] The second aspect of the present application discloses a method for preparing the above-mentioned novel skeleton diterpene alkaloid compound, which comprises the following extraction and separation steps:

[0009] S1, extracting the aconite root with a solvent by refluxing, combining the extracts and concentrating them to obtain an extract;

[0010] S2, dissolving the extract in an acidic solution, suspending, filtering and removing impurities to obtain a filtered acid aqueous solution; extracting the filtered acid aqueous solution with dichloromethane to obtain an acid aqueous solution A and an extract A;

[0011] The pH of the acid aqueous solution A is adjusted to be alkaline, and then extracted with dichloromethane to obtain an extract B;

[0012] S3, the extract B is sequentially subjected to column chromatography separation and high performance liquid chromatography separation to obtain the cleaved ring C having a new skeleton 20 Diterpene alkaloids;

[0013] Wherein, the column chromatography separation includes silica gel column chromatography, ODS column chromatography, and gel column chromatography performed in sequence.

[0014] Furthermore, the gel column chromatography is Sephadex LH-20 gel column chromatography.

[0015] Furthermore, in step S1, the solvent includes one or more of methanol, ethanol, acetone, chloroform and petroleum ether.

[0016] Further, in step S1, the solvent is 88-98V% ethanol aqueous solution;

[0017] The mass of solvent added is 8-10 times of that of aconite root; the number of reflux extractions is 2-4 times; each extraction takes 1-3 hours.

[0018] Furthermore, in step S2, the acidic solution is 0.1-1.0% dilute hydrochloric acid; the amount of the acidic solution added is 8-15 times the mass of the extract.

[0019] Furthermore, in step S2, a base is added to the acid aqueous solution A to adjust its pH to 8-11; preferably, pH=9.

[0020] Furthermore, based on the total volume of 100, in step S3, the silica gel column chromatography adopts gradient elution, and the mobile phase adopts a mixed solvent composed of solvent A and solvent B in a volume ratio of (0:100)-(100:0);

[0021] The solvent A is any one of dichloromethane, chloroform and petroleum ether;

[0022] The solvent B is any one of chloroform, acetone, ethyl acetate and methanol.

[0023] Preferably, the mobile phase is dichloromethane-methanol.

[0024] Further, step S3 includes:

[0025] S31, gradient eluting the extract B by silica gel column chromatography using a mobile phase of dichloromethane-methanol to obtain fraction C; when detected by silica gel thin layer chromatography, the Rf value corresponding to fraction C is 0.72-0.75;

[0026] S32, using methanol-water as the mobile phase to elute fraction C through ODS column chromatography to obtain fraction C3; when detected by silica gel thin layer chromatography, the Rf value corresponding to fraction C3 is 0.50-0.60;

[0027] S33, using methanol-water as a mobile phase to perform gel column chromatography on fraction C3 to obtain fraction M2; the Rf value of fraction M2 is 0.40-0.54;

[0028] S34. The fraction M2 is subjected to reverse phase high performance liquid chromatography with acetonitrile-water as the mobile phase to obtain a new skeleton diterpene alkaloid compound Carmiseconapline A with a structural formula as shown in formula (1); the retention time of the compound Carmiseconapline A is 8-13 min.

[0029] Furthermore, the retention time of compound Carmiseconapline A was 11 min.

[0030] Further, in step S31, the mobile phase of the silica gel column chromatography is dichloromethane-methanol in a volume ratio of (93:7)-(91:9). Preferably, the volume ratio of dichloromethane-methanol in the mobile phase is 92:8.

[0031] Furthermore, in step S32, the volume ratio of methanol-water in the ODS column chromatography mobile phase is (35:65)-(45:55); the mobile phase also contains 0.01-0.1% by volume of formic acid.

[0032] Preferably, in step S32, the volume ratio of methanol-water in the ODS column chromatography mobile phase is 40:60;

[0033] And / or, the volume proportion of formic acid in the mobile phase of the ODS column chromatography is 0.05%.

[0034] Furthermore, in step S33, the volume ratio of methanol-water in the gel column chromatography mobile phase is (25:75)-(35:65).

[0035] Preferably, in step S33, the volume ratio of the mobile phase methanol-water is 30:70.

[0036] Furthermore, in step S34, the high performance liquid chromatography uses a C18 chromatographic column, and the mobile phase uses acetonitrile-water with a volume ratio of (18:82)-(22:78), and the mobile phase also contains 0.01-0.5% trifluoroacetic acid by volume.

[0037] Furthermore, in step S34, the volume ratio of acetonitrile to water in the HPLC mobile phase is 20:80; and the volume proportion of trifluoroacetic acid in the mobile phase is 0.1%.

[0038] Among them, the tautomers or pharmaceutically acceptable salts of the novel skeleton diterpene alkaloid compounds also fall within the protection scope of the present invention.

[0039] The third aspect of the present application discloses a pharmaceutical composition comprising the novel skeleton diterpene alkaloid compound.

[0040] Furthermore, it also includes a pharmaceutically acceptable carrier or excipient.

[0041] That is, the cleaved ring C of the present invention is contained as an active ingredient 20 The pharmaceutical composition of diterpene alkaloid compounds and conventional pharmaceutical excipients or adjuvants or carriers is also included in the present invention.

[0042] Further, it also includes a synergist;

[0043] The synergist is at least one or more of fluoxetine, paroxetine, fluvoxamine, sertraline, citalopram, escitalopram, venlafaxine, duloxetine, mirtazapine, bupropion, agomelatine, trazodone, reboxetine, imipramine, amitriptyline, clomipramine, doxepin, maprotiline, moclobemide, Shugan Jieyu Capsule, St. John's wort extract, flupentixol and melitracen, etc.

[0044] Furthermore, the dosage form of the pharmaceutical composition is tablets, capsules, granules, oral liquid, granules, dropping pills, micropills, intravenous injections or intramuscular injections.

[0045] The present invention also provides the use of the novel skeleton diterpene alkaloid compound and / or the pharmaceutical composition in the preparation of antidepressant drugs.

[0046] The advantages and beneficial effects of the present invention are:

[0047] (1) The present invention provides a split ring C having a new skeleton 20 The invention discloses a diterpene alkaloid compound Carmiseconapline A, and further provides a method for extracting the split ring C from Radix Aconiti Lateralis with simple operation, good reproducibility and high extraction purity. 20 Methods for producing diterpene alkaloids;

[0048] (2) The split ring C with a new skeleton provided by the present invention 20 The diterpene alkaloid compound Carmiseconapline A showed significant antidepressant activity in the mouse behavioral despair model experiment. At a dose of 0.015 mg / kg, its efficacy was better than that of 10 mg / kg of the positive drug fluoxetine (a first-line clinical drug for the treatment of depression). At a dose of 0.06 mg / kg, its efficacy was twice that of 10 mg / kg of the positive drug fluoxetine, indicating that Carmiseconapline A has the potential to be used in the development of antidepressant drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The compound 1 prepared in Example 1 of the present invention 1 H NMR spectrum (600 MHz, D2O);

[0050] Figure 2 The compound 1 prepared in Example 1 of the present invention 13 C NMR spectrum (150 MHz, D2O);

[0051] Figure 3 This is the HMBC spectrum of compound 1 prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0052] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.

[0053] (I) Split ring C with a new skeleton 20 Preparation of diterpene alkaloids

[0054] Example 1

[0055] A split ring C with a new framework 20 The preparation method of diterpene alkaloid compounds comprises the following steps:

[0056] S1, take dried aconite root (about 200.5kg), reflux and extract three times with 95% ethanol aqueous solution with a mass of 10 times that of aconite root as solvent, each extraction is 2h, and the extracts are combined and concentrated to obtain an extract (about 6kg);

[0057] S2. Add the extract into 10 times the mass of 0.5% dilute hydrochloric acid (60L) to fully dissolve and suspend, filter to remove impurities, and extract the filtered acid aqueous solution with 1.5 times the volume of dichloromethane three times to obtain acidified dichloromethane extract A (about 2.5kg, discarded) and acid aqueous solution A (about 1.2kg).

[0058] The acid aqueous solution A was adjusted to pH = 9 with sodium hydroxide, and then extracted with dichloromethane to obtain extract B (about 500 g);

[0059] S3, the extract B is sequentially subjected to column chromatography separation and high performance liquid chromatography separation to obtain the cleaved ring C having a new skeleton 20 Diterpene alkaloid compounds; specifically including:

[0060] S31. Gradient elution of the extract B by silica gel column chromatography using dichloromethane-methanol to obtain fraction C; when detected by silica gel thin layer chromatography, the Rf value corresponding to fraction C is 0.72-0.75; the mobile phase of silica gel column chromatography is dichloromethane-methanol in a volume ratio of (93:7)-(91:9). Preferably, the volume ratio of dichloromethane-methanol in the mobile phase is 92:8.

[0061] It should be noted that during gradient elution, dichloromethane-methanol with a volume ratio of (93:7)-(91:9) can elute fraction C. Using silica gel thin layer chromatography, the fraction with an Rf value of 0.72-0.75 is fraction C. Among them, dichloromethane-methanol with a volume ratio of 92:8 has the highest elution efficiency. The principle of the "preferred volume ratio" described in the following gradient elution process is the same.

[0062] The specific operation includes: subjecting the extract B to silica gel column chromatography, using dichloromethane-methanol gradient elution with a volume ratio of 100:0, 95:5, 92:8, 90:10, 85:15, 75:25, 65:35, 50:50, 0:100, respectively, to obtain fractions 1-10, 11-20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, and a total of 90 fractions were collected and then identified by silica gel thin layer chromatography. According to the brick red spots shown by potassium iodide reagent, the Rf values ​​were observed to be 0.82-0.86 (fraction 1-10), 0.76-0.80 (fraction 11-20), 0.72-0.75 (fraction 21-30), 0.65-0.70 (fraction 31-40), 0.56-0.62 (fraction 41-50), 0.50-0.54 (fraction 51-60), 0.42-0.48 (fraction 61-70), 0.30-0.38 (fraction 71-80), 0.25-0.28 (fraction 81-90) and similar fractions were combined to obtain fractions A, B, C, D, E, F, G, H and I in turn; fractions 21-30 were combined to obtain fraction C.

[0063] According to the brick-red spots of potassium bismuth iodide in thin layer chromatography and the characteristic ultraviolet absorption (λ max =223 nm) Fraction C was selected for the next separation.

[0064] S32, using methanol-water as the mobile phase to elute fraction C through ODS column chromatography to obtain fraction C3; when detected by silica gel thin layer chromatography, the Rf value corresponding to fraction C3 is 0.50-0.60; the volume ratio of methanol-water in the ODS column chromatography mobile phase is (35:65)-(45:55); the mobile phase also contains 0.01-0.1% formic acid by volume. Preferably, the volume ratio of methanol-water is 40:60; the volume ratio of formic acid in the mobile phase is 0.05%.

[0065] The specific operation includes: fraction C is subjected to ODS column chromatography, and gradient elution is performed using methanol-water (containing 0.05% formic acid by volume) with a volume ratio of 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0 to obtain fractions 1-5, 6-15, 16-25, 26-35, 36-45, 46-50, 51-55, 56-60, 61-68 in turn, and a total of 68 fractions are collected. According to the analysis and identification, the Rf values ​​of the brick-red spots shown by the potassium iodide reagent were observed to be 0.35-0.44 (fractions 1-5), 0.45-0.49 (fractions 6-15), 0.50-0.60 (fractions 16-25), 0.61-0.69 (fractions 26-35), 0.70-0.74 (fractions 36-55), 0.75-0.79 (fractions 56-60), and 0.80-0.89 (fractions 61-68). Similar fractions were combined into 7 fractions C1 to C7. Fractions 16-25 were combined to obtain fraction C3.

[0066] According to the brick-red spots of potassium bismuth iodide reagent in thin layer chromatography and the characteristic ultraviolet absorption (λ max =223 nm) fraction C3 was selected for the next separation.

[0067] S33, using a mobile phase of methanol-water to perform gel column chromatography on fraction C3 to obtain fraction M2; the Rf value of fraction M2 is 0.40-0.54; the volume ratio of the mobile phase of the gel column chromatography, methanol-water, is (25:75)-(35:65). Preferably, the volume ratio of methanol-water is 30:70.

[0068] The specific operation includes: fraction C3 is subjected to Sephadex LH-20 column chromatography, and a methanol-water gradient elution is performed with a volume ratio of 0:100, 30:70, 70:30, and 100:0, and fractions 1-6, 7-14, 15-20, and 21-29 are collected in sequence. After a total of 29 fractions are collected, the fractions are identified by silica gel thin layer chromatography, and the Rf values ​​observed based on the brick red spots revealed by potassium iodide reagent are 0.30-0.39 (fractions 1-6), 0.40-0.54 (fractions 7-14), 0.55-0.61 (fractions 15-20), and 0.62-0.85 (fractions 21-29), and similar fractions are combined to obtain four fractions M1 to M4 in sequence. Fractions 7-14 are combined to obtain fraction M2.

[0069] According to the brick-red spots revealed by potassium bismuth iodide reagent and the characteristic ultraviolet absorption (λ max =223 nm) fraction M2 was selected for the next separation.

[0070] S34, fraction M2 is subjected to reverse phase high performance liquid chromatography, with acetonitrile-water as the mobile phase, to obtain Carmiseconapline A, a new skeleton diterpene alkaloid compound with a structural formula as shown in formula (1); the retention time of compound Carmiseconapline A is 8-13min. The high performance liquid chromatography uses a C18 chromatographic column, and the mobile phase uses acetonitrile-water with a volume ratio of (18:82)-(22:78), and the mobile phase also contains 0.01-0.5% trifluoroacetic acid by volume. Preferably, the acetonitrile-water volume ratio is 20:80; the volume proportion of trifluoroacetic acid in the mobile phase is 0.1%.

[0071] The specific operation includes: using HPLC method, using acetonitrile-water (containing 0.1% trifluoroacetic acid in volume ratio) with a volume ratio of 20:80 as the mobile phase, using C 18 The chromatographic column was prepared in fraction M2 to obtain the split ring C 20 Diterpene alkaloids 1 (compound Carmiseconapline A) (t R =11 min, purity 98%).

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

[0073] Physical properties: White amorphous powder, easily soluble in methanol.

[0074] High resolution mass spectrometry (HR-ESI-MS) m / z 388.2113 ([M+H] + The calculated value is 388.2118), combined with 1 HNMR Figure 1 ),13 CNMR spectrum ( Figure 2 ), and its molecular formula is determined to be C 22 H 29 O5N, its unsaturation is calculated to be 9.

[0075] 1 HNMR (600MHz, D2O) spectrum ( Figure 1 ) shows that it contains a group of hydrogen signals on the terminal double bond [δ H 5.44 (1H, m), 5.41 (1H, d, J = 1.9 Hz)], two hydrogen signals on the oxygen-linked methine [δ H 5.08 (1H, dd, J = 10.5, 7.5 Hz), 4.33 (1H, s)], the hydrogen signals on the three azine methylene groups [δ H 3.72 (1H, t, J = 11.9 Hz), 3.28 (2H, m), 3.20 (2H, overlapped), 3.14 (1H, m)] and two methyl signals [δ H 1.35(3H,overlap),1.34(3H,s)], and in δ H There are 3 methine and 4 methylene signals between 3.52-1.53.

[0076] 13 CNMR (150MHz, D2O) spectrum ( Figure 2 ) shows a total of 22 carbon signals, including 6 sp 2 Hybridized carbon [δ C 179.8 (C-12), 176.1 (C-11), 171.1 (C-10), 150.2 (C-16), 124.2 (C-19) and 115.0 (C-17)], 5-linked oxygen / nitrogen sp 3 Hybridized carbon [δ C 82.8 (C-15), 80.2 (C-1), 64.9 (C-19), 60.9 (C-20), 57.6 (C-21)] and 11 aliphatic sp 3 Hybridized carbon.

[0077] 1 H NMR, 13 The signal assignments of C NMR are shown in Table 1.

[0078] Table 1 1 H NMR, 13 C NMR signal assignment δ (ppm)

[0079]

[0080] Combination 1H NMR and 13 C NMR spectrum, it is speculated that compound 1 may contain a terminal double bond structure, a conjugated double bond and two carbonyl signals, and the remaining 5 unsaturations are speculated to be occupied by 5 rings. In addition, it also contains 5 carbon signals connected to oxygen / nitrogen and 2 methyl groups. Combining the above information, it is speculated that compound 1 may be a highly oxidative ring-cleaved C 20 Type diterpene alkaloids.

[0081] In the HMBC spectrum ( Figure 3 ), the HMBC correlation peaks of H2-21 with C-19 and C-20 and their displacement values ​​indicate that C-19, C-20, and C-21 are all connected to nitrogen atoms. The correlation peaks of H2-6 with C-20 can be inferred that C-6 is associated with C-20. At the same time, considering that C-20 is a methylene group, it can be inferred that this represents an unprecedented C-10 / C-20 bond breakage. 20 The carbon skeleton of diterpene alkaloids. The connection mode of spiro ring C was established through the correlation peaks of H2-17 with C-15 and C-16, the correlation peaks of H-15 with C-7, C-13 and C-14, and the correlation peaks of H-13 and H2-14 with carboxyl carbon C-12. In addition, the position of the quaternary carbonyl carbon between C-9 and C-10 was determined through the HMBC correlation peaks of H-1, H-5, H2-14 with C-9 and H-1, H-5 with C-10. Combined with the molecular formula and chemical shift values ​​of C-9, C-10 and C-11, the lactone ring D must be formed between the oxygen-containing C-1 and the carboxyl C-11 in 1.

[0082] In summary, the structure of the new compound 1 (Carmiseconapline A) was determined as follows:

[0083]

[0084] Comparative Example 1

[0085] The preparation method is the same as that of Example 1, except that in step S34, the aqueous solution used to prepare the mobile phase for separating the fraction M2 by HPLC does not contain trifluoroacetic acid.

[0086] The compound prepared in Comparative Example 1 had severe peak tailing during the preparation process. Liquid phase analysis after preparation revealed that it contained a large amount of impurities. The purity of the prepared compound 1 was less than 60%.

[0087] Comparative Example 2

[0088] The preparation method is the same as that of Example 1, with the only difference being that in step S32, when fraction C is eluted by ODS column chromatography, methanol-water (without formic acid) with an initial volume ratio of 40:60 is used.

[0089] Comparative Example 2: This compound could not be detected during the subsequent fraction preparation process:

[0090] The results showed that compound 1 could not be prepared.

[0091] (II) Verification experiment

[0092] Experimental Example 1: Carmiseconapline A mouse behavioral despair model experiment (the gold standard for antidepressant drug screening)

[0093] 1. Experimental Materials and Instruments

[0094] Fluoxetine hydrochloride was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; higenamine was purchased from Shanghai Standard Standard Technical Service Co., Ltd.; total alkaloids from Aconitum carmichaelii, Aconapelsulfonine B and Carmiseconapline A were all homemade in the laboratory.

[0095] ZIL-2 type mouse self-activity box (Shanghai Xinman Science and Education Equipment Co., Ltd.); YLS-18A type mouse tail suspension instrument (Anhui Zhenghua Biological Instrument Equipment Co., Ltd.); electronic balance (Beijing Sartorius Scientific Instrument Co., Ltd.).

[0096] 2. Experimental Animals

[0097] ICR mice, male, weighing 18-22g, SPF grade, provided by Weitong Lihua, animal license number: SYXK (Beijing) 2023-0001. Animals were raised in an environment with a temperature of 23±2℃, a humidity of 50%±10%, and a lighting time of 12h per day (lighting from 7:00 to 19:00). The experiments were carried out after the animals were raised for 3 days. They were fasted for 6h before the start of the behavioral experiment and had free access to water.

[0098] 3. Experimental Methods

[0099] 64 ICR male mice were divided into 8 groups in descending order of the number of spontaneous activities according to the animal screening, namely, blank group (same volume of normal saline), positive drug fluoxetine hydrochloride group (10 mg / kg), aconite total alkaloid group (10 mg / kg), higenamine group (10 mg / kg), aconite B group (0.06 mg / kg), carmiseconapline A low-dose group (0.015 mg / kg), carmiseconapline A medium-dose group (0.03 mg / kg), carmiseconapline A high-dose group (0.06 mg / kg), 8 mice in each group, numbered respectively. All drugs were added to normal saline to form a suspension, and each group was gavaged once a day, with a volume of 0.1 ml / 10 g body weight. After 3 consecutive days of administration, spontaneous activity experiments were performed on the 4th day, and tail suspension experiments were performed on the 5th day.

[0100] 4. Experimental Results

[0101] As shown in Table 2, compared with the blank group, the low, medium and high dose groups of Carmiseconapline A (0.015, 0.03, 0.06 mg / kg) can significantly shorten the immobility time of mice in the tail suspension test. At the low dose (0.015 mg / kg), Carmiseconapline A showed an antidepressant effect superior to the positive drugs fluoxetine (10 mg / kg, the first-line chemical drug for clinical treatment of depression), total alkaloids of aconite (10 mg / kg), higenamine (10 mg / kg) and Aconicarmisulfonine B (0.06 mg / kg); at the high dose (0.06 mg / kg), the antidepressant effect of Carmiseconapline A was twice that of fluoxetine (10 mg / kg).

[0102] Table 2 Effects of each group on the immobility time of mice in the tail suspension test

[0103]

[0104] (mean ± standard error, compared with the blank group, * P<0.05, *** P<0.001)

[0105] As shown in Table 3, in the open field test, each group had no significant effect on the spontaneous activity of mice compared with the blank group, indicating that the effect of each group on the immobility time of mice in the tail suspension test was not related to neural excitability.

[0106] Table 3 Effects of each group on the number of horizontal grids crossed by mice in the open field test

[0107]

[0108] In summary, the present invention has a novel skeleton split ring C 20 The diterpene alkaloid Carmiseconapline A has significant antidepressant activity, which is significantly better than the positive drug fluoxetine and can be used as a drug precursor for the treatment of depression.

[0109] Application Example 1

[0110] The application example of the present invention discloses a capsule preparation using Carmiseconapline A as a raw material drug, and its components are as follows:

[0111]

[0112] The specific preparation process is as follows:

[0113] Carmiseconapline A, starch and sodium metabisulfite were mixed evenly, anhydrous ethanol was added to make a soft material, the mixture was passed through a 24-mesh sieve to make granules, the mixture was dried, magnesium stearate was added, the mixture was mixed evenly and the mixture was put into capsules.

[0114] Application Example 2

[0115] The application example of the present invention discloses a granule preparation using the compound Carmiseconapline A as a raw material drug, and the components thereof are as follows:

[0116] Carmiseconapline A4.0mg

[0117] Starch 6.0g

[0118] Sodium bisulfite 0.2g

[0119] Magnesium stearate 0.2g

[0120] Anhydrous ethanol

[0121] Makes 100 bags.

[0122] The specific preparation process is as follows:

[0123] Carmiseconapline A is mixed with starch and sodium bisulfite, and then anhydrous ethanol is added to make a soft material. The mixture is passed through a 24-mesh sieve to form granules, which are dried, and magnesium stearate is added. The mixture is mixed and bagged.

[0124] Application Example 3

[0125] The application example of the present invention discloses an oral liquid with the compound Carmiseconapline A as the raw material medicine, and its components are as follows:

[0126]

[0127] The specific preparation process is as follows:

[0128] After the above components are mixed evenly, they can be packaged using a conventional oral liquid preparation method.

[0129] Application Example 4

[0130] The application example of the present invention discloses an injection with the compound Carmiseconapline A as the raw material drug, and its components are as follows:

[0131] Carmiseconapline A 4.0mg

[0132] Vitamin C 0.2g

[0133] Sodium chloride 6.0g

[0134] Sodium bicarbonate 0.5g

[0135] Water for injection 1000.0mL

[0136] Made into 100 pieces.

[0137] The specific preparation process is as follows:

[0138] After the above components are mixed evenly, 100 vials can be obtained using a conventional injection preparation method.

[0139] Application Example 5

[0140] The application example of the present invention discloses a tablet with compounds Carmiseconapline A and fluoxetine as raw materials, and its components are as follows:

[0141] Carmiseconapline A 3.5mg

[0142] Fluoxetine 20.0mg

[0143] Hydroxypropyl methylcellulose 18.0g

[0144] Talc 0.4g

[0145] Lactose 0.2g

[0146] Magnesium stearate 0.2g

[0147] Anhydrous ethanol

[0148] Makes 100 pieces.

[0149] The specific preparation process is as follows:

[0150] Carmiseconapline A, fluoxetine, hydroxypropyl methylcellulose, talc, lactose and magnesium stearate were mixed evenly, anhydrous ethanol was added to make a soft material, the mixture was passed through a 24-mesh sieve to form granules, dried, magnesium stearate was added, mixed evenly and tableted.

[0151] Application Example 6

[0152] The application example of the present invention discloses a capsule preparation using compounds Carmiseconapline A and Luyoutai as raw materials, and the components thereof are as follows:

[0153] Carmiseconapline A 3.5mg

[0154] Luyoutai 300.0mg

[0155] Starch 6.0g

[0156] Sodium metabisulfite 0.2g

[0157] Magnesium stearate 0.2g

[0158] Anhydrous ethanol

[0159] Makes 100 tablets.

[0160] The specific preparation process is as follows:

[0161] Carmiseconapline A, Luyoutai, starch and sodium metabisulfite were mixed, anhydrous ethanol was added to make a soft material, the mixture was passed through a 24-mesh sieve to make granules, the granules were dried, magnesium stearate was added, the mixture was mixed, and the granules were filled into capsules.

[0162] Application Example 7

[0163] The application example of the present invention discloses an injection with compound Carmiseconapline A and paroxetine as raw materials, and its components are as follows:

[0164] Carmiseconapline A 5.0mg

[0165] Paroxetine 20.0mg

[0166] Vitamin C 0.2g

[0167] Sodium chloride 6.0g

[0168] Sodium bicarbonate 0.5g

[0169] Water for injection 1000.0mL

[0170] Made into 100 pieces.

[0171] The specific preparation process is as follows:

[0172] After the above components are mixed evenly, 100 vials can be obtained using a conventional injection preparation method.

[0173] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A novel skeleton diterpene alkaloid compound, characterized in that: It has the structure shown in formula (1): （1）。 2. The novel skeleton diterpene alkaloid compound according to claim 1, characterized in that: It is extracted and separated from Aconite root.

3. A method for preparing the compound according to claim 1 or 2, characterized in that: The method comprises the following extraction and separation steps: S1, extracting the aconite root with a solvent by refluxing, combining the extracts and concentrating them to obtain an extract; S2, dissolving the extract in an acidic solution, suspending, filtering and removing impurities to obtain a filtered acid aqueous solution; extracting the filtered acid aqueous solution with dichloromethane to obtain an acid aqueous solution A and an extract A; The pH of the acid aqueous solution A is adjusted to be alkaline, and then extracted with dichloromethane to obtain an extract B; S3, the extract B is subjected to column chromatography separation and high performance liquid chromatography separation in sequence to obtain the new skeleton diterpene alkaloid compound; Wherein, the column chromatography separation includes silica gel column chromatography, ODS column chromatography, and gel column chromatography performed in sequence.

4. The preparation method according to claim 3, characterized in that: In step S1, the solvent is one or more of methanol, ethanol, acetone, chloroform and petroleum ether.

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

6. The preparation method according to claim 3, characterized in that: In step S2, the acidic solution is 0.1-1.0% dilute hydrochloric acid; the amount of the acidic solution added is 8-15 times the mass of the extract.

7. The preparation method according to claim 3, characterized in that: In step S2, a base is added to the acid aqueous solution A to adjust the pH thereof to 8-11.

8. The preparation method according to claim 3, characterized in that: Step S3 includes: S31, gradient eluting the extract B by silica gel column chromatography using dichloromethane-methanol to obtain fraction C; when detected by silica gel thin layer chromatography, the Rf value corresponding to fraction C is 0.72-0.75; S32, using methanol-water as the mobile phase to elute fraction C through ODS column chromatography to obtain fraction C3; when detected by silica gel thin layer chromatography, the Rf value corresponding to fraction C3 is 0.50-0.60; S33, using methanol-water as a mobile phase to perform gel column chromatography on fraction C3 to obtain fraction M2; the Rf value of fraction M2 is 0.40-0.54; S34. The fraction M2 was subjected to reverse phase high performance liquid chromatography with acetonitrile-water as the mobile phase to obtain a new skeleton diterpene alkaloid compound Carmiseconapline A with a structural formula as shown in formula (1); the retention time of the compound Carmiseconapline A was 8-13 min.

9. The preparation method according to claim 8, characterized in that: The retention time of compound Carmiseconapline A is 11 min.

10. The preparation method according to claim 8, characterized in that: In step S31, the mobile phase of the silica gel column chromatography is dichloromethane-methanol in a volume ratio of (93:7)-(91:9).

11. The preparation method according to claim 8, characterized in that: In step S31, the volume ratio of the mobile phase dichloromethane-methanol is 92:

8.

12. The preparation method according to claim 8, characterized in that: In step S32, the volume ratio of methanol-water in the ODS column chromatography mobile phase is (35:65)-(45:55); the mobile phase also contains 0.01-0.1% by volume of formic acid.

13. The preparation method according to claim 12, characterized in that: In step S32, the volume ratio of methanol-water in the ODS column chromatography mobile phase is 40:60; And / or, the volume proportion of formic acid in the mobile phase of the ODS column chromatography is 0.05%.

14. The preparation method according to claim 8, characterized in that: In step S33, the volume ratio of methanol-water in the mobile phase of the gel column chromatography is (25:75)-(35:65).

15. The preparation method according to claim 8, characterized in that: In step S33, the volume ratio of the mobile phase methanol-water is 30:

70.

16. The preparation method according to claim 8, characterized in that: In step S34, the high performance liquid chromatography uses a C18 chromatographic column, and the mobile phase uses acetonitrile-water with a volume ratio of (18:82)-(22:78), and the mobile phase also contains 0.01-0.5% trifluoroacetic acid by volume.

17. The preparation method according to claim 16, characterized in that: In step S34, the volume ratio of acetonitrile to water in the HPLC mobile phase is 20:80; and the volume proportion of trifluoroacetic acid in the mobile phase is 0.1%.

18. A pharmaceutical composition, characterized in that The invention comprises the novel skeleton diterpene alkaloid compound as described in claim 1 or 2.

19. The pharmaceutical composition according to claim 18, characterized in that Also included are pharmaceutically acceptable carriers or excipients.

20. The pharmaceutical composition according to claim 18, characterized in that Also included are synergists; The synergist is any one or more of the following substances: fluoxetine, paroxetine, fluvoxamine, sertraline, citalopram, escitalopram, venlafaxine, duloxetine, mirtazapine, bupropion, agomelatine, trazodone, reboxetine, imipramine, amitriptyline, clomipramine, doxepin, maprotiline, moclobemide, Shugan Jieyu Capsule, St. John's wort extract, flupentixol and melitracen.

21. The pharmaceutical composition according to claim 18, characterized in that The dosage form of the pharmaceutical composition is tablets, capsules, granules, oral liquid, granules, dropping pills or micropills.

22. Use of the novel skeleton diterpene alkaloid compound according to claim 1 or 2 and / or the pharmaceutical composition according to any one of claims 18 to 21 in the preparation of antidepressant drugs.

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