Novel lucentine-type diterpene alkaloid compound and its preparation method and application

Abstract

The present invention relates to the field of medical technology, and discloses a novel chalcogenin-type diterpene alkaloid compound and its preparation method and application. 20 The delphinidin-type diterpenoid alkaloids carmaloidline C, carmaloidline D, and aconialoidline C all exhibit significant analgesic effects. Carmaloidline C exhibits the strongest analgesic activity, surpassing the positive-acting drug morphine and significantly outperforming the positive-acting drug ibuprofen. Carmaloidline D and aconialoidline C exhibited greater analgesic activity than the positive-acting drug ibuprofen. These drugs could be used to develop analgesics for various acute and chronic pain conditions.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a novel chalcogenin-type diterpene alkaloid compound, a preparation method and application thereof. Background Art

[0002] Pain is an essential protective function of the human body, often accompanied by alterations in multiple systems, including metabolism, endocrine function, respiratory function, circulatory function, and psychology. It is a serious public health concern and one of the most common clinical symptoms. According to incomplete statistics, over 300 million people worldwide suffer from chronic pain, with nearly 50% of patients experiencing chronic pain, sometimes chronically, due to a lack of effective treatment. Nearly 100 million people in my country suffer from chronic pain. Pain has transcended the concept of a symptom and has become a serious condition requiring universal attention. In 2001, the Asia-Pacific Pain Forum declared that "the elimination of pain is a fundamental right of patients."

[0003] Current clinical treatments for various acute and chronic pain conditions often suffer from toxic side effects, low safety, and adverse reactions such as addiction and dependence with long-term use. Natural medicinal plants are not only widely distributed, diverse in species, and possess complex and diverse chemical compositions, but many natural remedies have been used to treat pain for thousands of years. Modern experimental research on these natural medicinal plants can provide guidance for the search for new, highly effective, low-toxic, and non-addictive analgesics.

[0004] The Chinese herbal medicine Aconite is the lateral root of the plant Aconitum carmichaelii Debeaux of the Ranunculaceae family. In order to find new analgesic drug candidates, the inventors isolated a new type of chrysanthemum-type C from Aconite. 20 Diterpene alkaloid compounds Carmaloidline C, Carmaloidline D and Aconialoidline C were identified, and it was found that Carmaloidline C exhibited significant analgesic activity in the acetic acid writhing test and hot plate test in mice, which was superior to the positive drug morphine and significantly superior to ibuprofen. The analgesic activity of Carmaloidline D and Aconialoidline C was superior to the positive drug ibuprofen, and they can be used as drugs for treating pain. Summary of the Invention

[0005] The purpose of the present invention is to overcome the defects in the prior art and provide a novel chalcogenin-type diterpene alkaloid compound and a preparation method, a pharmaceutical composition and an application thereof.

[0006] To achieve the above-mentioned object, the technical solution of the present invention is to provide a novel class of chalcogenin-type diterpene alkaloid compounds having the structures shown in formula (1), formula (2) and / or formula (3):

[0007]

[0008] For the sake of simplicity, the novel carmaloidine-type diterpene alkaloid compounds having formulas (1) to (3) are referred to as compounds Carmaloidline C, Carmaloidline D and Aconialoidline C, respectively.

[0009] The present invention provides a method for preparing the novel lucentine-type diterpene alkaloid compound. The novel lucentine-type diterpene alkaloid compound is extracted and separated from aconite root.

[0010] Furthermore, the following extraction and separation steps are included:

[0011] S1. Extracting the aconite root with a solvent under reflux, combining the extracts and concentrating them to obtain an extract;

[0012] S2. Dissolving the extract obtained in step S1 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;

[0013] Adjusting the pH value of the acid aqueous solution A to alkaline, and then extracting with dichloromethane to obtain extract B;

[0014] S3, the extract B is sequentially subjected to column chromatography and high performance liquid chromatography to obtain the novel chalcogenide-type diterpene alkaloid compound;

[0015] Wherein, the column chromatography separation includes silica gel column chromatography, ODS column chromatography, and gel column chromatography performed in sequence.

[0016] Preferably, the gel column chromatography is Sephadex LH-20 gel column chromatography.

[0017] Furthermore, in step S1, the solvent is an 88-98V% ethanol aqueous solution;

[0018] The mass of the solvent added is 8-10 times that of the aconite root; the number of reflux extractions is 2-4 times; each extraction takes 1-3 hours.

[0019] Furthermore, in step S2, the acidic solution is 0.1-1.0% dilute hydrochloric acid; and the amount of the acidic solution added is 8-15 times the mass of the extract.

[0020] Furthermore, in step S2, the pH value of the acid aqueous solution A is adjusted to 8-11 by adding a base; preferably, the pH value is 9.

[0021] Furthermore, taking the total volume as 100,

[0022] In step S3, the silica gel column chromatography adopts gradient elution, and the mobile phase adopts a mixed solvent consisting of solvent A and solvent B in a volume ratio of (0:100) to (100:0);

[0023] The solvent A is any one of dichloromethane, chloroform and petroleum ether;

[0024] The solvent B is any one of chloroform, acetone, ethyl acetate and methanol.

[0025] Furthermore, step S3 includes:

[0026] S31, gradient eluting the extract B using a mobile phase through silica gel column chromatography to obtain a fraction C; when identified by silica gel thin layer chromatography, the Rf value corresponding to the fraction C is 0.72-0.75;

[0027] S32. Fraction C was eluted by ODS column chromatography using a mobile phase of methanol-water to obtain fraction C5; when detected by silica gel thin layer chromatography, the Rf value corresponding to fraction C5 was 0.70-0.74;

[0028] S33. Fraction C5 is subjected to gel column chromatography using a mobile phase of methanol-water to obtain fraction N3; the Rf value of fraction N3 is 0.66-0.76;

[0029] S34, fraction N3 was subjected to reverse phase high performance liquid chromatography with acetonitrile-water as the mobile phase to obtain a novel chalcogenide-type diterpene alkaloid compound Carmaloidline C having the structural formula shown in formula (1); the retention time of the compound Carmaloidline C was 40-45 min;

[0030] A novel chalcogenide-type diterpene alkaloid compound Carmaloidline D having a structural formula as shown in formula (2) is obtained; the retention time of the compound Carmaloidline D is 46-50 min;

[0031] A novel chalcogenin-type diterpene alkaloid compound Aconialoidline C having a structural formula as shown in formula (3) was obtained; the retention time of the compound Aconialoidline C was 35-40 min.

[0032] Preferably, the retention time of compound Aconialoidline C is 39 min;

[0033] The retention time of compound Carmaloidline C was 45 min;

[0034] The retention time of compound Carmaloidline D is 47 min.

[0035] Preferably, the mobile phase in step S31 is dichloromethane-methanol.

[0036] Furthermore, in step S31, the mobile phase of the silica gel column chromatography is dichloromethane-methanol in a volume ratio of (93:7)-(91:9).

[0037] Preferably, in step S31, the volume ratio of the mobile phase dichloromethane to methanol is 92:8.

[0038] Furthermore, in step S32, the volume ratio of methanol-water in the ODS column chromatography mobile phase is (55:45)-(65:35); and the mobile phase contains 0.01-0.1% by volume of formic acid.

[0039] Preferably, in step S32, the volume ratio of methanol to water in the ODS column chromatography mobile phase is 60:40;

[0040] And / or, the volume proportion of formic acid in the mobile phase of the ODS column chromatography is 0.05%.

[0041] Furthermore, in step S33, the volume ratio of methanol-water in the gel column chromatography mobile phase is (65:35)-(75:25).

[0042] Preferably, in step S33, the volume ratio of the mobile phase methanol to water is 70:30.

[0043] Furthermore, in step S34, the high performance liquid chromatography adopts C 18 The chromatographic column uses a mobile phase of acetonitrile-water with a volume ratio of (15:85)-(20:80), and the mobile phase also contains 0.01-0.5% trifluoroacetic acid by volume.

[0044] Furthermore, in step S34, the volume ratio of acetonitrile to water in the high performance liquid chromatography mobile phase is 17:83; and the volume proportion of trifluoroacetic acid in the mobile phase is 0.1%.

[0045] Among them, the tautomers or pharmaceutically acceptable salts of the novel lucite-type diterpene alkaloid compounds also fall within the protection scope of the present invention.

[0046] The third aspect of the present application discloses a pharmaceutical composition comprising the novel lucentine-type diterpene alkaloid compound.

[0047] Furthermore, it also includes a pharmaceutically acceptable carrier or excipient.

[0048] That is, the pharmaceutical composition containing the delphinidinine-type diterpene alkaloid compound of the present invention as an active ingredient and conventional pharmaceutical excipients, adjuvants or carriers is also included in the present invention.

[0049] Further, it also includes a synergist;

[0050] The synergist is one or more of the following substances:

[0051] Aspirin, ibuprofen, ketoprofen, nabucolyn, meloxicam, methotrexate, elodinil, felodipine, Tylenol, acetaminophen, morphine, MyxoContin, meperidine, fentanyl, oxycodone, hydrocodone, oxycodone, codeine, acetaminophen, amitriptyline, oxaspirin, roxicam, fluoxetine, gabapentin, pregabalin, carbamazepine, lidocaine, bupivacaine, meloxicam, phenacetin, methylhydrazine, rocuronium, ketorolac tromethamine, lornoxicam, celecoxib, naltrexone, naloxone, methadone, dihydroetorphine, tramadol, pentazocine, buprenorphine, and bucinnazine.

[0052] Furthermore, the dosage form of the pharmaceutical composition is tablets, capsules, granules, oral liquid, granules, dripping pills, micropills, intravenous injection or intramuscular injection.

[0053] The present invention also provides the use of the novel lucite-type diterpene alkaloid compound or the pharmaceutical composition in the preparation of a drug for preventing or treating pain.

[0054] The advantages and beneficial effects of the present invention are:

[0055] (1) The present invention provides a class of delphinidin-type C 20 Diterpenoid alkaloid compounds, and further provide a method for extracting a novel delphinidin-type C from aconite root with simple operation, good reproducibility and high extraction purity. 20 Methods for producing diterpene alkaloids;

[0056] (2) The experimental results show that the delphinidin-type diterpene alkaloids provided by the present invention all have analgesic effects. Among them, the compound carmaloidline C has a significant analgesic effect, which is better than the positive drug morphine and significantly better than the positive drug ibuprofen. The analgesic activity of carmaloidline D and aconialoidline C is better than the positive drug ibuprofen, and can be used to develop analgesics for various acute or chronic pains. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Compound 1 obtained in Example 1 of the present invention 1H NMR spectrum (400 MHz, CDCl3);

[0058] Figure 2 Compound 1 obtained in Example 1 of the present invention 13 C NMR spectrum (100 MHz, CDCl3);

[0059] Figure 3 This is the HMBC spectrum of compound 1 prepared in Example 1 of the present invention;

[0060] Figure 4 The NOESY spectrum of compound 1 prepared in Example 1 of the present invention;

[0061] Figure 5 Compound 2 obtained in Example 1 of the present invention 1 H NMR spectrum (600 MHz, CDCl3);

[0062] Figure 6 Compound 2 obtained in Example 1 of the present invention 13 C NMR spectrum (150 MHz, CDCl3);

[0063] Figure 7 This is the HMBC spectrum of compound 2 prepared in Example 1 of the present invention;

[0064] Figure 8 The compound 3 obtained in Example 1 of the present invention 1 H NMR spectrum (400 MHz, DMSO-d6);

[0065] Figure 9 The compound 3 obtained in Example 1 of the present invention 13 C NMR spectrum (100 MHz, DMSO-d6);

[0066] Figure 10 This is the HMBC spectrum of compound 3 prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0067] The following embodiments of the present invention are further described in conjunction with the accompanying drawings and examples. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

[0068] (1) Preparation of chloroquine-type diterpene alkaloid compounds

[0069] Example 1:

[0070] This embodiment discloses a method for preparing a novel lucentine-type diterpene alkaloid compound, comprising the following steps:

[0071] S1, take aconite root (about 200.5kg), use 95% ethanol aqueous solution with a mass 10 times that of aconite root as a solvent for reflux extraction three times, each extraction for 2h, and then concentrate the combined extracts to obtain an extract (about 6kg);

[0072] S2. The extract was added to 10 times the mass of 0.5% dilute hydrochloric acid (60 L) and fully dissolved and suspended, and then filtered to remove impurities. The filtered acid aqueous solution was extracted three times with 1.5 times the volume of dichloromethane to obtain acidified dichloromethane extract A (about 2.5 kg, discarded) and acid aqueous solution A (about 1.2 kg);

[0073] The acid aqueous solution A was adjusted to pH = 9 with sodium hydroxide, and then extracted with dichloromethane to obtain a dichloromethane extract, namely extract B (about 500 g).

[0074] S3. The extract B is sequentially subjected to column chromatography separation and high performance liquid chromatography separation to obtain the novel chalcogenin-type diterpene alkaloid compound.

[0075] Step S3 specifically includes:

[0076] S31, gradient eluting the extract B using a mobile phase through silica gel column chromatography to obtain a fraction C; when identified by silica gel thin layer chromatography, the Rf value corresponding to the fraction C is 0.72-0.75;

[0077] The mobile phase of silica gel column chromatography is a mixed solvent composed of solvent A and solvent B in a volume ratio of (0:100)-(100:0); solvent A is any one of dichloromethane, chloroform and petroleum ether; solvent B is any one of chloroform, acetone, ethyl acetate and methanol.

[0078] The mobile phase for silica gel column chromatography is preferably dichloromethane-methanol in a volume ratio of (93:7) to (91:9). Further preferably, the volume ratio of dichloromethane-methanol is 92:8.

[0079] It should be noted that during gradient elution, fraction C can be eluted using dichloromethane-methanol volume ratios ranging from (93:7) to (91:9). Using silica gel thin-layer chromatography, the fraction with an Rf value of 0.72-0.75 is fraction C. Among them, a dichloromethane-methanol volume ratio of 92:8 has the highest elution efficiency. The principles described in the "preferred" gradient elution process above and below are the same.

[0080] The specific operation includes: extract B is subjected 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, 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, a total of 90 fractions were collected and then identified by silica gel thin layer chromatography. According to the brick red spots revealed by potassium bismuth iodide reagent, the Rf values ​​were observed to be 0.82-0.86 (fraction Similar fractions were combined to obtain fractions A, B, C, D, E, F, G, H and I in sequence; fractions 21-30 were combined to obtain fraction C.

[0081] According to the brick-red spots of potassium bismuth iodide in thin layer chromatography and the characteristic ultraviolet absorption (λ max =235nm) Fraction C was selected for the next separation.

[0082] S32. Fraction C was eluted by ODS column chromatography using methanol-water as the mobile phase to obtain fraction C5; when identified by silica gel thin layer chromatography, the Rf value corresponding to fraction C5 was 0.70-0.74.

[0083] The volume ratio of methanol to water in the ODS column chromatography mobile phase is (55:45)-(65:35); the mobile phase also contains 0.01-0.1% by volume of formic acid. Preferably, the volume ratio of methanol to water in the ODS column chromatography mobile phase is 60:40; the volume ratio of formic acid in the mobile phase is 0.05%.

[0084] The specific operation includes: fraction C is subjected to ODS column chromatography, and a gradient elution is performed using methanol-water (containing 0.05% formic acid by volume of the mobile phase) with a volume ratio of 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 100:0, to obtain fractions 1-5, 6-15, 16-25, 26-35, 36-45, 46-50, 51-55, 56-60, and 61-68, for a total of 68 fractions. Thin layer chromatography analysis revealed brick-red spots revealed by potassium bismuth iodide reagent, and the Rf values ​​were 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 36-45), 0.75-0.79 (fractions 46-60), and 0.80-0.89 (fractions 61-68). Similar fractions were combined to form seven fractions, C1 to C7. Fractions 36-45 were combined to give fraction C5.

[0085] According to the brick-red spots of potassium bismuth iodide in thin layer chromatography and the characteristic ultraviolet absorption (λ max =235nm) and fraction C5 was selected for the next separation.

[0086] S33. Using the mobile phase of methanol-water, fraction C5 is subjected to gel column chromatography to obtain fraction N3; the Rf value of fraction N3 is 0.66-0.76; the volume ratio of the mobile phase methanol-water is (65:35)-(75:25); preferably 70:30.

[0087] The specific operation includes: subjecting fraction C5 to Sephadex LH-20 gel column chromatography, using a methanol-water gradient elution with a volume ratio of 0:100, 30:70, 70:30, and 100:0, and collecting fractions 1-5, 6-15, 16-23, and 24-33 in sequence. After collecting a total of 33 fractions, they are identified by silica gel thin layer chromatography. According to the brick-red spots revealed by potassium bismuth iodide reagent, the Rf values ​​are observed to be 0.35-0.52 (fractions 1-5), 0.53-0.65 (fractions 6-15), 0.66-0.76 (fractions 16-23), and 0.77-0.85 (fractions 24-33), respectively. Similar fractions are combined to form four fractions N1 to N4; fractions 16-23 are combined to obtain fraction N3.

[0088] According to the brick-red spots of potassium bismuth iodide in thin layer chromatography and the characteristic ultraviolet absorption (λ max =235nm) fraction N3 was used for the next separation.

[0089] S34. Fraction N3 is subjected to reverse-phase high performance liquid chromatography with acetonitrile-water as the mobile phase to obtain Carmaloidline C, a novel chalcogenin-type diterpene alkaloid compound having the structural formula shown in formula (1); the retention time of compound Carmaloidline C is 40-45 min; Carmaloidline D, a novel chalcogenin-type diterpene alkaloid compound having the structural formula shown in formula (2); the retention time of compound Carmaloidline D is 46-50 min; Aconialoidline C, a novel chalcogenin-type diterpene alkaloid compound having the structural formula shown in formula (3); the retention time of compound Aconialoidline C is 35-40 min.

[0090] The high performance liquid chromatography adopts a C18 chromatographic column, and the mobile phase adopts acetonitrile-water with a volume ratio of (15:85)-(20:80), and the mobile phase also contains 0.01-0.5% trifluoroacetic acid by volume.

[0091] Preferably, the HPLC method is used, with acetonitrile-water (containing 0.1% trifluoroacetic acid by volume of the total volume of acetonitrile-water) in a volume ratio of 17:83 as the mobile phase, and a C18 chromatographic column is used to prepare the delphinidinyl-type C in fraction N3. 20 Diterpene alkaloid compound 3 (compound Aconialoidline C) (t R =39min, purity 98%), compound 1 (compound Carmaloidline C) (t R =45min, purity 99%), compound 2 (compound Carmaloidline D) (t R =47 min, purity 98%).

[0092] The physical properties and test data of Compound 1 prepared in Example 1 are as follows:

[0093] Physical properties: White amorphous powder, easily soluble in methanol.

[0094] High resolution mass spectrometry (HR-ESI-MS) m / z 498.2842 ([M+H] + The calculated value is 498.2850), combined with 1 H NMR ( Figure 1 ), 13 C NMR spectrum ( Figure 2 ), and its molecular formula is determined to be C 29 H 39 NO6, its unsaturation is calculated to be 11.

[0095] 1 H NMR (400 MHz, CDCl3) spectrum ( Figure 1 ) shows that there is a group of hydrogen signals on the monosubstituted benzene ring in the low field region [δ H 8.02 (2H, d, J = 7.8 Hz), 7.56 (1H, t, J = 7.4 Hz), 7.43 (2H, dd, J = 7.8, 7.4 Hz)], 10 hydrogen signals on oxygen / nitrogen carbon [δ H 4.78 (2H, brs), 4.62 (1H, d, J = 9.6 Hz), 4.15 (1H, s), 3.98 (1H, s), 3.71 (1H, s), 2.55 (1H, d, J = 8.8 Hz), 2.45 (1H, overlapped), 2.25 (1H, d, J = 8.8 Hz), 1.81 (1H, overlapped)]; there are two methyl hydrogen signals in the high field region [δ H 1.08(3H, overlapped)and 0.72(3H,s)].

[0096] 13 C NMR (100 MHz, CDCl3) spectrum ( Figure 2 ) showed a total of 29 carbon signals, including 2 methyl carbon signals (δ C 26.1,13.5), 5 methylene carbon signals (δ C 38.6, 31.4, 27.7, 23.6, 20.7), 4 methine carbon signals (δ C 59.7, 52.6, 45.6, 42.7), 8 connected oxygen / nitrogen-carbon signals (δ C 85.3,79.5,71.5,70.9,70.5,67.4,57.1,51.3), 1 carbonyl carbon signal (δ C 1667.2), carbon signals on the six benzene rings (δ C 133.3, 130.2, 129.8, 129.8, 128.6, 128.6) and the remaining three quaternary carbon signals (δ C 50.9,43.5,33.9).

[0097] 1 H NMR, 13 The signal assignments of C NMR are shown in Table 1.

[0098] Table 1 Compound 1 1 H NMR, 13 C NMR signal assignment δ (ppm)

[0099]

[0100] Combine 1 H NMR and 13 C NMR spectrum, it is speculated that compound 1 may contain a benzoyl fragment, and the remaining 6 unsaturations are assumed to be occupied by 6 rings. In addition, it also contains 8 carbon signals connected to oxygen / nitrogen and 2 methyl groups. Combined with the above information, it is speculated that compound 1 may be a C 20 Type diterpene alkaloids.

[0101] In the HMBC spectrum ( Figure 3 ), H-17(δ H 4.78) and C-7′(δ C 167.2), thus confirming that the benzoyl fragment is attached to the oxygen at C-17; in addition, H-22 (δ H 1.08) and C-21(δ C The long-range correlation between the two groups indicated that the N-terminal was connected to a methylene group. Compared with the known compound Lassiocarpine, the planar structure of the compound was confirmed.

[0102] In NOESY spectrum ( Figure 4 ), H-11(δ H 4.62) and H-13(δ H 1.48),H-11(δ H 4.62) and H-1(δ H 4.15),H-17(δ H 4.78) and H-12(δ H 1.81), it can be determined that the orientation of H-11, H-1 and 16-OH is α.

[0103] In summary, the structure of the new compound 1 (Carmaloidline C) was determined as follows:

[0104]

[0105] The physical properties and test data of compound 2 prepared in Example 1 are as follows:

[0106] Physical properties: White amorphous powder, easily soluble in methanol.

[0107] High resolution mass spectrometry (HR-ESI-MS) m / z 498.2840 ([M+H] + The calculated value is 498.2850), combined with 1 H NMR ( Figure 5 ), 13 C NMR spectrum ( Figure 6 ), and its molecular formula is determined to be C29 H 39 NO6, its unsaturation is calculated to be 11.

[0108] 1 H NMR (600 MHz, CDCl3) spectrum ( Figure 5 ) shows that there is a group of hydrogen signals on the monosubstituted benzene ring in the low field region [δ H 8.02 (2H, d, J = 7.6 Hz), 7.61 (1H, t, J = 7.4 Hz), 7.48 (2H, dd, J = 7.6, 7.4 Hz)], 10 groups of hydrogen signals on oxygen / nitrogen and carbon [δ H 5.19 (1H, s), 4.77 (1H, d, J = 5.8 Hz), 4.36 (1H, J = 10.5 Hz), 4.33 (1H, s), 4.19 (1H, brs), 3.87 (1H, d, J = 10.5 Hz), 3.19 (1H, d, J = 13.0 Hz), 3.09 (1H, brs), 2.92 (1H, brs), 2.38 (1H, d, J = 13.0 Hz)]; there are two methyl hydrogen signals in the high field region [δ H 1.33(3H,t,J=7.2Hz),0.74(3H,s)].

[0109] 13 C NMR (150 MHz, CDCl3) spectrum ( Figure 6 ) showed a total of 29 carbon signals, including 2 methyl carbon signals (δ C 25.6,9.8), 5 methylene carbon signals (δ C 37.0, 29.2, 27.8, 23.1, 19.7), 4 methine carbon signals (δ C 53.4, 50.3, 43.9, 42.6), 8 connected oxygen / nitrogen-carbon signals (δ C 84.3,78.9,69.5,67.5,67.4,65.4,57.2,54.4), 1 carbonyl carbon signal (δ C 166.5), carbon signals on the six benzene rings (δ C 133.8, 129.8, 129.7, 129.7, 128.9, 128.9) and the remaining three quaternary carbon signals (δ C 52.4,42.8,34.6).

[0110] 1 H NMR, 13 The signal assignments of C NMR are shown in Table 2.

[0111] Table 2 Compound 2 1 H NMR,13 C NMR signal assignment δ (ppm)

[0112]

[0113] Combine 1 H NMR and 13 C NMR spectrum, it is speculated that compound 1 may contain a benzoyl fragment, and the remaining 6 unsaturations are assumed to be occupied by 6 rings. In addition, it also contains 7 carbon signals connected to oxygen / nitrogen and 2 methyl groups. Combined with the above information, it is speculated that compound 2 may be a C 20 Type diterpene alkaloids.

[0114] In the HMBC spectrum ( Figure 7 ), H-15(δ H 5.19) and C-16 (δ C 78.9), C-8(δ C 42.8), C-7′(δ C 166.5), thus confirming that the benzoyl fragment is attached to the oxygen on C-15; H-15 (δ H 5.19) and C-17 (δ C 67.4) and the chemical shift of H-17 confirmed that C-17 is connected to C-16 and C-17 is connected to hydroxyl group; in addition, H-22 (δ H 1.33) and C-21(δ C The long-range correlation between 54.4) proves that the bond to N is a methylene group.

[0115] In summary, the structure of the new compound 2 (Carmaloidline D) was determined as follows:

[0116]

[0117] The physical properties and test data of compound 3 prepared in Example 1 are as follows:

[0118] Physical properties: White amorphous powder, easily soluble in methanol.

[0119] High resolution mass spectrometry (HR-ESI-MS) m / z 538.2798 ([M+H] + The calculated value is 538.2799), combined with 1 H NMR ( Figure 8 ), 13 C NMR spectrum ( Figure 9 ), and its molecular formula is determined to be C 31 H 39 O7N, its unsaturation is calculated to be 13.

[0120] 1 HNMR (400 MHz, DMSO-d6) spectrum ( Figure 8 ) in δ H 8.51 (1H, s, H-19) There is one olefin proton signal. H There are five aromatic proton signals at 8.06 (2H, d, J = 7.2 Hz, H-2′, 6′), 7.65 (1H, t, J = 7.4 Hz, H-4′), and 7.51 (2H, d, J = 7.6 Hz, H-3′, 5′). H 4.89 (1H, s, H-15), 4.52 (1H, overlapped, H-20), 4.48 (1H, overlapped, H-11), 4.44 (2H, overlapped, H-17), 4.12 (1H, t, J = 8.4 Hz, H-1), 4.00 (1H, dq, J = 13.9, 7.0 Hz, H-21a), 3.91 (1H, dq, J = 13.9, 7.0 Hz, H-21b). There are 8 proton signals on nitrogen or oxygen carbon. H There are three methyl proton signals at 2.00 (3H, s, 2″), 1.38 (3H, m, H-22), and 1.16 (3H, s, H-18).

[0121] 13 CNMR (100 MHz, DMSO-d6) spectrum ( Figure 9 ), there are 31 carbon signals in total. Including one carbon-nitrogen double bond carbon signal: δ C 182.9 (C-19); 2 ester carbonyl carbon signals: δ C 169.9 (C-1″), 165.8 (C-7′); carbon signals on the six benzene rings δ C 133.0 (C-4′), 130.2 (C-1′), 129.5 (C-2′, 6′), 128.5 (C-3′, 5′); 5 oxygen-linked carbon signals: δ C 83.4 (C-15), 75.5 (C-16), 68.9 (C-17), 68.1 (C-11), 66.4 (C-1); 2 nitrogen-carbon signals: δ C 69.8 (C-20), 55.9 (C-21); 3 methyl carbon signals: δ C 21.1 (C-2″), 19.9 (C-18), 13.4 (C-22); 5 methylene carbon signals: δ C34.4 (C-3), 30.0 (C-2), 26.4 (C-14), 24.6 (C-6), 19.4 (C-13); 4 methine carbon signals δ C 45.7 (C-5), 47.7 (C-7), 51.3 (C-9), 44.1 (C-12) and three quaternary carbon signals: δ C 43.9(C-4),43.1(C-8),50.9(C-10).

[0122] Combined with the above data, it is speculated that compound 3 may contain 1 benzoyloxy fragment, 1 acetoxy fragment and 1 imine fragment. The remaining 6 unsaturations are assumed to be occupied by 6 rings. In addition, it contains 7 carbon signals connected to oxygen / nitrogen and 3 methyl signals. It is speculated that compound 3 may be a photodelphinidin-type C 20 -Diterpene alkaloids.

[0123] 1 H NMR, 13 The signal assignments of C NMR are shown in Table 3 below.

[0124] Table 3 Compound 3 1 H NMR, 13 C NMR signal assignment δ (ppm)

[0125]

[0126] In the HMBC spectrum ( Figure 10 ), H-15(δ H 4.89) and H-2″(δ H 2.00) are all the same as C-1″(δ C 169.9) showed long-range correlation, which confirmed that the acetoxy group was connected to the C-15 position; H-17 (δ H 4.44) and H-2′ / 6′(δ H 8.06) are all related to C-7′(δ C 165.8) showed a long-range correlation, confirming that C-17 was connected to a benzoyloxy group. Literature comparison revealed that it was similar to 15-O-acetyllassiocarpine, with only a difference between C-19 and N. Further analysis of H-19 (δ H 8.51) and C-4(δ C 43.9),C-5(δ C 45.7),C-18(δ C 19.9),C-21(δ C 55.9),C-20(δ C69.8) The presence of long-range correlation and chemical shifts of H-19 and C-19 confirmed that C-19 and N formed an imine structure.

[0127] In summary, the structure of the new compound 3 (Aconialoidline C) was determined as follows:

[0128]

[0129] Comparative Example 1:

[0130] The preparation method is the same as that of Example 1, with the only difference being that in step S34, the aqueous solution used in the mobile phase for separating fraction N3 by HPLC does not contain trifluoroacetic acid.

[0131] 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 compounds 1, 2 and 3 was less than 20%.

[0132] Comparative Example 2:

[0133] The preparation method is the same as that of Example 1, with the only difference being that in step S32, when fraction C is subjected to ODS column chromatography, methanol-water (without formic acid) with an initial volume ratio of 20:80 is used for elution.

[0134] In Comparative Example 2, compounds 1, 2, and 3 could not be detected during subsequent fraction preparation:

[0135] The results showed that compounds 1, 2 and 3 could not be prepared.

[0136] (2) Verification experiment

[0137] Experimental Example 1: Acetic acid writhing test of compounds Carmaloidline C, Carmaloidline D and Aconialoidline C

[0138] Test substances: morphine, ibuprofen, total alkaloids of aconite root, aconiticatisulfonine B, carmaloidline C prepared in Example 1, carmaloidline D, and aconialoidline C.

[0139] Experimental animals: ICR mice, male, weighing 23-27 g, SPF grade, provided by Weitonglihua, animal license number: SYXK (Beijing) 2023-0001.

[0140] Reagents and consumables: glacial acetic acid (Beijing Inokai Technology Co., Ltd.), normal saline (Beijing Pulizhicheng Biotechnology Co., Ltd.), ibuprofen (Shanghai Yuanye Biotechnology Co., Ltd.), total alkaloids from aconite root and aconiticatisulfonine B were homemade in the laboratory, 12# gavage needle (Shanghai Titan Technology Co., Ltd.), timer (Jiangsu KeyGen Biotechnology Co., Ltd.), 1mL syringe (Beijing Sypulus Technology Co., Ltd.).

[0141] (2) Experimental methods

[0142] Grouping: After 2 days of adaptive feeding, ICR mice were randomly divided into 11 groups according to their body weight, with 8 mice in each group, namely model group, ibuprofen group (20 mg / kg), morphine group (0.3 mg / kg), aconite total alkaloids group (10 mg / kg), acononicatisulfonine B group (0.3 mg / kg), carmaloidline C low-dose group (0.03 mg / kg), carmaloidline C high-dose group (0.3 mg / kg), carmaloidline D low-dose group (0.03 mg / kg), carmaloidline D high-dose group (0.3 mg / kg), aconialoidline C low-dose group (0.03 mg / kg), and aconialoidline C high-dose group (0.3 mg / kg).

[0143] Dosage and administration method: The mice in the model group were gavaged with normal saline solution; the mice in the ibuprofen group were gavaged with ibuprofen in normal saline solution at a dose of 0.1 mL / 10 g; the mice in the morphine group were gavaged with morphine in normal saline solution at a dose of 0.1 mL / 10 g; the mice in the aconite total alkaloid group were gavaged with aconite total alkaloid solution in normal saline solution at a dose of 0.1 mL / 10 g; the mice in the aconialoidline B group were gavaged with aconialoidline B in normal saline solution at a dose of 0.1 mL / 10 g; the mice in the low- and high-dose carmaloidline C groups were gavaged with carmaloidline C in normal saline solution at a dose of 0.1 mL / 10 g; the mice in the low- and high-dose carmaloidline D groups were gavaged with carmaloidline D in normal saline solution at a dose of 0.1 mL / 10 g; and the mice in the low- and high-dose aconialoidline C groups were gavaged with aconialoidline C in normal saline solution at a dose of 0.1 mL / 10 g. Models were established 1 hour after gavage administration according to the above method.

[0144] Modeling: One hour after oral gavage, the mice in the model group, ibuprofen group, morphine group, aconite total alkaloid group, aconiticatisulfonine B group, carmaloidline C group, carmaloidline D group, and aconialoidline C group were intraperitoneally injected with 0.7% glacial acetic acid solution (0.1 mL / 10 g). The number of writhing reactions of the mice within 10 minutes after injection was counted, and the inhibition rate of writhing reactions was calculated. The time of the first writhing reaction was recorded as the pain threshold.

[0145] Inhibition rate of writhing times (%) = [(writhing times in model group - writhing times in drug-treated group) / writhing times in model group] × 100

[0146] (3) Experimental results

[0147] As shown in Table 4, compared with the model group, the number of writhing events in mice was significantly reduced after administration of low and high doses (0.03 and 0.3 mg / kg) of carmaloidline C, carmaloidline D, and aconialoidline C. The inhibitory rate of low-dose carmaloidline C on the number of writhing events in mice was significantly higher than that of the positive drugs ibuprofen (20 mg / kg), total alkaloids of aconite root (10 mg / kg), and aconialoidline B (0.3 mg / kg). The inhibitory rate of high-dose carmaloidline C on the number of writhing events in mice was higher than that of the positive drug morphine (0.3 mg / kg) at the same dose. The inhibitory rates of high-dose carmaloidline D and aconialoidline C on the number of writhing events in mice were higher than those of the positive drugs ibuprofen (20 mg / kg), total alkaloids of aconite root (10 mg / kg), and aconialoidline B (0.3 mg / kg).

[0148] Table 4 Inhibition rate of writhing times in mice after intraperitoneal injection of 0.7% acetic acid solution

[0149]

[0150]

[0151] (Compared with the model group, *** P < 0.001)

[0152] As shown in Table 5, compared with the model group, low and high doses (0.03 and 0.3 mg / kg) of carmaloidline C, carmaloidline D, and aconialoidline C significantly prolonged the pain latency of mice (P<0.001). The low dose of carmaloidline C significantly outperformed the active drugs ibuprofen (20 mg / kg), total alkaloids from aconite root (10 mg / kg), and aconialoidine B (0.3 mg / kg) in prolonging the pain latency of mice. The high dose of carmaloidline C significantly outperformed the active drug morphine (0.3 mg / kg) at the same dose. The high doses of carmaloidline D and aconialoidline C significantly outperformed the active drugs ibuprofen (20 mg / kg), total alkaloids from aconite root (10 mg / kg), and aconialoidine B (0.3 mg / kg) in prolonging the pain latency of mice.

[0153] Table 5 Pain latency in mice after intraperitoneal injection of 0.7% acetic acid solution

[0154]

[0155]

[0156] (Compared with the model group, *** P < 0.001)

[0157] In summary, photodelphinidin type C 20 The diterpenoid alkaloid compounds carmaloidline C, carmaloidline D, and aconialoidline C exhibited significant analgesic activity in the acetic acid writhing test, and at a dose of 0.3 mg / kg, they were superior to the positive drugs ibuprofen, total aconite alkaloids, and aconialoidine B. Among them, carmaloidline C had the strongest analgesic activity, surpassing the positive drugs ibuprofen, total aconite alkaloids, and aconialoidine B at a dose of 0.03 mg / kg, and surpassing the positive drug morphine at a dose of 0.3 mg / kg.

[0158] Experimental Example 2: Hot Plate Experiment of Carmaloidline C

[0159] (1) Experimental materials

[0160] Test substances: ibuprofen, morphine, total alkaloids from Aconite root, Aconicatisulfonine B and Carmaloidline C prepared in Example 1.

[0161] Experimental animals: ICR mice, male, weighing 23-27 g, SPF grade, provided by Weitonglihua, animal license number: SYXK (Beijing) 2023-0001.

[0162] Reagents and consumables: Normal saline (Beijing Pulizhicheng Biotechnology Co., Ltd.), ibuprofen (Shanghai Yuanye Biotechnology Co., Ltd.), total alkaloids from aconite root and aconiticatisulfonine B were homemade in the laboratory, 12# gavage needle (Shanghai Titan Technology Co., Ltd.), timer (Jiangsu Keygen Biotechnology Co., Ltd.), 1 mL syringe (Beijing Sypurus Technology Co., Ltd.).

[0163] (2) Experimental methods

[0164] Grouping: After 2 days of adaptive feeding, ICR mice were randomly divided into 7 groups according to body weight, with 8 mice in each group, namely, blank group, ibuprofen group (20 mg / kg), morphine group (0.3 mg / kg), aconite total alkaloid group (10 mg / kg), aconiticosulfonine B group (0.3 mg / kg), low-dose carmaloidline C group (0.03 mg / kg), and high-dose carmaloidline C group (0.3 mg / kg).

[0165] Dosage and administration method: The mice in the blank group were gavaged with normal saline solution; the mice in the ibuprofen group were gavaged with normal saline solution of ibuprofen at a dose of 0.1 mL / 10 g; the mice in the morphine group were gavaged with normal saline solution of morphine at a dose of 0.1 mL / 10 g; the mice in the aconite total alkaloid group were gavaged with normal saline solution of aconite total alkaloids at a dose of 0.1 mL / 10 g; the mice in the aconite total alkaloid group were gavaged with normal saline solution of aconite total alkaloids at a dose of 0.1 mL / 10 g; the mice in the carmaloidline C low-dose and high-dose groups were gavaged with normal saline solution of carmaloidline C at a dose of 0.1 mL / 10 g.

[0166] Measurement and record: The temperature of the hot plate was adjusted to 55.5°C, and the pain threshold of the mice was measured and recorded at 0.5, 1, 2, 3, 4, and 5 h after gavage, and the number of times the mice lifted their feet 0.5 h after gavage was recorded.

[0167] (3) Experimental results

[0168] As shown in Table 6, compared with the blank group, both the low- and high-dose carmaloidline C groups (0.03 and 0.3 mg / kg) significantly reduced the number of paw lifts in mice 0.5 h after oral gavage (P < 0.001). Low-dose carmaloidline C was significantly more effective than the active agents ibuprofen (20 mg / kg), aconite total alkaloids (0.3 mg / kg), and aconiticatisulfonine B (0.3 mg / kg) in reducing the number of paw lifts in mice. High-dose carmaloidline C was comparable to the active agent morphine at the same dose in reducing the number of paw lifts in mice.

[0169] Table 6 Number of foot lifts after 0.5 h of oral gavage in mice hot plate experiment

[0170]

[0171]

[0172] (Compared with the blank group, *** P < 0.001)

[0173] As shown in Table 7, compared with the blank control group, both the low- and high-dose carmaloidline C groups (0.03 and 0.3 mg / kg) significantly increased the pain threshold of mice 0.5, 1, and 2 hours after oral gavage. Low-dose carmaloidline C significantly outperformed the positive drug groups ibuprofen (20 mg / kg), aconite total alkaloids (10 mg / kg), and aconiticarmisulfonine B (0.3 mg / kg) in increasing the pain threshold of mice. High-dose carmaloidline C also outperformed the positive drug group morphine at the same dose in increasing the pain threshold of mice.

[0174] Table 7 Pain latency in hot plate test in mice

[0175]

[0176] (Compared with the blank group, ** P<0.01, *** P < 0.001)

[0177] In summary, the present invention's delphinidin-type C 20 The diterpene alkaloid compound Carmaloidline C showed significant analgesic activity in the hot plate test, which was better than the positive drug morphine, and significantly better than the positive drugs ibuprofen, total alkaloids of aconite and aconiticatisulfonine B.

[0178] The present invention discloses a type of delphinidin-type C 20The diterpenoid alkaloid compounds Carmaloidline C, Carmaloidline D and Aconialoidline C have analgesic activity superior to the clinical first-line analgesics morphine and ibuprofen, and can be used as drug prodrugs for the treatment of pain.

[0179] (III) Application of Carmaloidline C, Carmaloidline D, and Aconialoidline C

[0180] Application Example 1:

[0181] The present invention discloses an application example of a capsule containing Carmaloidline C as a raw material drug, the components of which are as follows:

[0182]

[0183] The specific preparation process is as follows:

[0184] Carmaloidline C, starch and sodium metabisulfite were mixed evenly, anhydrous ethanol was added to form a soft material, the material was passed through a 24-mesh sieve, granulated, dried, magnesium stearate was added, mixed evenly and filled into capsules.

[0185] Application Example 2:

[0186] The present invention discloses an application example of a granule preparation using the compound Carmaloidline C as a raw material drug, and its components are as follows:

[0187]

[0188] The specific preparation process is as follows:

[0189] Carmaloidline C is mixed with starch and sodium bisulfite, and then anhydrous ethanol is added to form a soft material. The mixture is passed through a 24-mesh sieve to form granules, dried, and magnesium stearate is added. The mixture is mixed and bagged.

[0190] Application Example 3:

[0191] The present invention discloses an oral solution using the compound Carmaloidline C as a raw material drug, and its components are as follows:

[0192]

[0193] The specific preparation process is as follows:

[0194] After the above components are mixed evenly, they can be packaged using a conventional oral liquid preparation method.

[0195] Application Example 4:

[0196] The application example of the present invention discloses an injection with the compound Carmaloidline C as the raw material drug, and its components are as follows:

[0197]

[0198] The specific preparation process is as follows:

[0199] After the above components are mixed evenly, 100 injections can be obtained using conventional injection preparation methods.

[0200] Application Example 5:

[0201] The present invention discloses an application example of a tablet comprising carmaloidline C and aspirin as raw materials, the components of which are as follows:

[0202]

[0203] The specific preparation process is as follows:

[0204] Carmaloidline C, aspirin, hydroxypropyl methylcellulose, talc, lactose, and magnesium stearate are mixed evenly, anhydrous ethanol is added to form a soft material, the material is passed through a 24-mesh sieve, granulated, dried, magnesium stearate is added, mixed evenly, and tableted.

[0205] Application Example 6:

[0206] The present invention discloses an application example of a capsule preparation using compound Carmaloidline C and ibuprofen as raw materials, and its components are as follows:

[0207]

[0208] The specific preparation process is as follows:

[0209] Carmaloidline C, ibuprofen, starch and sodium metabisulfite are mixed, anhydrous ethanol is added to form a soft material, the mixture is passed through a 24-mesh sieve, granulated, dried, magnesium stearate is added, mixed, and filled into capsules.

[0210] Application Example 7:

[0211] The application example of the present invention discloses an injection with compounds Carmaloidline C and Meloxicam as raw materials, and its components are as follows:

[0212]

[0213] The specific preparation process is as follows:

[0214] After the above components are mixed evenly, 100 injections can be obtained using conventional injection preparation methods.

[0215] Application Example 8:

[0216] The present invention discloses an application example of a capsule preparation using Carmaloidline D as a raw material drug, and its components are as follows:

[0217]

[0218] The specific preparation process is as follows:

[0219] Carmaloidline D, starch and sodium metabisulfite were mixed evenly, anhydrous ethanol was added to form a soft material, the mixture was passed through a 24-mesh sieve, granulated, dried, magnesium stearate was added, mixed evenly and filled into capsules.

[0220] Application Example 9:

[0221] The present invention discloses an application example of a granule preparation using the compound Carmaloidline D as a raw material drug, and its components are as follows:

[0222]

[0223] The specific preparation process is as follows:

[0224] Carmaloidline D was mixed with starch and sodium bisulfite, and then anhydrous ethanol was added to form a soft material. The mixture was passed through a 24-mesh sieve to form granules. The granules were dried, magnesium stearate was added, the mixture was mixed, and the mixture was bagged.

[0225] Application Example 10:

[0226] The application example of the present invention discloses an oral solution using the compound Carmaloidline D as the raw material drug, and its components are as follows:

[0227]

[0228] The specific preparation process is as follows:

[0229] After the above components are mixed evenly, they can be packaged using a conventional oral liquid preparation method.

[0230] Application Example 11:

[0231] The application example of the present invention discloses an injection with the compound Carmaloidline D as the raw material drug, and its components are as follows:

[0232]

[0233]

[0234] The specific preparation process is as follows:

[0235] After the above components are mixed evenly, 100 injections can be obtained using conventional injection preparation methods.

[0236] Application Example 12:

[0237] The application example of the present invention discloses a tablet with compound Carmaloidline D and aspirin as raw materials, and its components are as follows:

[0238]

[0239] The specific preparation process is as follows:

[0240] Carmaloidline D, aspirin, hydroxypropyl methylcellulose, talc, lactose, and magnesium stearate were mixed evenly, anhydrous ethanol was added to form a soft material, the material was passed through a 24-mesh sieve, granulated, dried, magnesium stearate was added, mixed evenly, and tableted.

[0241] Application Example 13:

[0242] The present invention discloses an application example of a capsule preparation using the compound Carmaloidline D and ibuprofen as raw materials, and its components are as follows:

[0243]

[0244]

[0245] The specific preparation process is as follows:

[0246] Carmaloidline D, ibuprofen, starch and sodium metabisulfite are mixed, anhydrous ethanol is added to form a soft material, the material is passed through a 24-mesh sieve, granulated, dried, magnesium stearate is added, the mixture is mixed, and the mixture is filled into capsules.

[0247] Application Example 14:

[0248] The application example of the present invention discloses an injection with the compound Carmaloidline D and meloxicam as raw materials, and its components are as follows:

[0249]

[0250] The specific preparation process is as follows:

[0251] After the above components are mixed evenly, 100 injections can be obtained using conventional injection preparation methods.

[0252] Application Example 15:

[0253] The present invention discloses an application example of a capsule preparation using Aconialoidline C as a raw material drug, and its components are as follows:

[0254]

[0255]

[0256] The specific preparation process is as follows:

[0257] Aconialoidline C, starch and sodium metabisulfite were mixed evenly, anhydrous ethanol was added to form a soft material, the material was passed through a 24-mesh sieve, granulated, dried, magnesium stearate was added, mixed evenly and filled into capsules.

[0258] Application Example 16:

[0259] The present invention discloses an application example of a granule preparation using the compound Aconialoidline C as a raw material drug, and its components are as follows:

[0260]

[0261] The specific preparation process is as follows:

[0262] Aconialoidline C is mixed with starch and sodium bisulfite, and then anhydrous ethanol is added to form a soft material. The mixture is passed through a 24-mesh sieve to form granules, which are then dried. Magnesium stearate is added, the mixture is mixed, and the mixture is bagged.

[0263] Application Example 17:

[0264] The application example of the present invention discloses an oral solution using the compound Aconialoidline C as the raw material drug, and its components are as follows:

[0265]

[0266]

[0267] The specific preparation process is as follows:

[0268] After the above components are mixed evenly, they can be packaged using a conventional oral liquid preparation method.

[0269] Application Example 18:

[0270] The application example of the present invention discloses an injection with the compound Aconialoidline C as the raw material drug, and its components are as follows:

[0271]

[0272] The specific preparation process is as follows:

[0273] After the above components are mixed evenly, 100 injections can be obtained using conventional injection preparation methods.

[0274] Application Example 19:

[0275] The present invention discloses an application example of a tablet comprising aconialoidline C and aspirin as raw materials, the components of which are as follows:

[0276]

[0277] The specific preparation process is as follows:

[0278] Aconialoidline C, aspirin, hydroxypropyl methylcellulose, talc, lactose, and magnesium stearate were mixed evenly, anhydrous ethanol was added to form a soft material, the material was passed through a 24-mesh sieve, granulated, dried, magnesium stearate was added, mixed evenly, and tableted.

[0279] Application Example 20:

[0280] The present invention discloses an application example of a capsule preparation using the compound Aconialoidline C and ibuprofen as raw materials, and its components are as follows:

[0281]

[0282] The specific preparation process is as follows:

[0283] Aconialoidline C, ibuprofen, starch and sodium metabisulfite are mixed, anhydrous ethanol is added to form a soft material, the material is passed through a 24-mesh sieve, granulated, dried, magnesium stearate is added, mixed, and filled into capsules.

[0284] Application Example 21:

[0285] The present invention discloses an injection using the compound Aconialoidline C and meloxicam as raw materials, and its components are as follows:

[0286]

[0287] The specific preparation process is as follows:

[0288] After the above components are mixed evenly, 100 injections are obtained using a conventional injection preparation method. The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the technical principles of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a delphinidin-type diterpene alkaloid compound, characterized in that: The diterpene alkaloid compounds have the structure shown in formula (1), formula (2) or formula (3): The novel delphinidin-type diterpenoid alkaloid compound is extracted and separated from aconite root; The preparation method comprises the following extraction and separation steps: S1, extracting the aconite root with a solvent by refluxing, combining the extracts and concentrating to obtain an extract; the solvent is 88-98V% ethanol aqueous solution; S2. Dissolving the extract obtained in step S1 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 acidic solution is 0.1-1.0% dilute hydrochloric acid; Adjusting the pH value of the acid aqueous solution A to alkaline, and then extracting with dichloromethane to obtain extract B; S3, sequentially subjecting the extract B to column chromatography and high performance liquid chromatography separation to obtain the novel delphinidin-type diterpene alkaloid compound; Wherein, the column chromatography separation includes silica gel column chromatography, ODS column chromatography, and gel column chromatography performed in sequence; Step S3 includes: S31, gradient eluting the extract B through silica gel column chromatography using a mobile phase to obtain a fraction C; when identified by silica gel thin layer chromatography, the Rf value corresponding to the fraction C is 0.72-0.75; the mobile phase is dichloromethane-methanol; S32. Fraction C is eluted by ODS column chromatography using a mobile phase of methanol-water to obtain fraction C5; when detected by silica gel thin layer chromatography, the Rf value corresponding to fraction C5 is 0.70-0.74; the volume ratio of methanol-water in the ODS column chromatography mobile phase is (55:45)-(65:35); the mobile phase contains 0.01-0.1% by volume of formic acid; S33. Fraction C5 is subjected to gel column chromatography using a mobile phase of methanol-water to obtain fraction N3; the Rf value of fraction N3 is 0.66-0.76; the volume ratio of methanol-water in the mobile phase of the gel column chromatography is (65:35)-(75:25); S34, fraction N3 was subjected to reverse phase high performance liquid chromatography with acetonitrile-water as the mobile phase to obtain a novel delphinidin-type diterpene alkaloid compound Carmaloidline C having the structural formula shown in formula (1); the retention time of compound Carmaloidline C was 40-45 min; A novel delphinidin-type diterpene alkaloid compound Carmaloidline D having a structural formula as shown in formula (2) is obtained; the retention time of the compound Carmaloidline D is 46-50 min; A novel delphinidin-type diterpene alkaloid compound Aconialoidline C having a structural formula as shown in formula (3) is obtained; the retention time of the compound Aconialoidline C is 35-40 min; The high performance liquid chromatography (HPLC) was carried out using C 18 The chromatographic column uses a mobile phase of acetonitrile-water with a volume ratio of (15:85)-(20:80), and the mobile phase also contains 0.01-0.5% trifluoroacetic acid by volume.

2. The preparation method according to claim 1, wherein In step S1, the mass of the solvent added is 8-10 times that of the aconite root; the number of reflux extractions is 2-4 times; and each extraction takes 1-3 hours.

3. The preparation method according to claim 1, wherein In step S2, the amount of the acidic solution added is 8-15 times the mass of the extract.

4. The preparation method according to claim 1, wherein In step S2, a base is added to the acid aqueous solution A to adjust the pH value thereof to 8-11.

5. The preparation method according to claim 1, wherein The retention time of compound Aconialoidline C was 39 min; The retention time of compound Carmaloidline C was 45 min; The retention time of compound Carmaloidline D is 47 min.

6. The preparation method according to claim 1, wherein In step S31 , the mobile phase for silica gel column chromatography is dichloromethane-methanol in a volume ratio of (93:7)-(91:9).

7. The preparation method according to claim 1, wherein In step S31, the volume ratio of the mobile phase, dichloromethane:methanol, is 92:

8.

8. The preparation method according to claim 1, wherein In step S32, the volume ratio of methanol to water in the ODS column chromatography mobile phase is 60:40; The volume proportion of formic acid in the mobile phase of the ODS column chromatography is 0.05%.

9. The preparation method according to claim 1, wherein In step S33, the volume ratio of the mobile phase methanol to water is 70:

30.

10. The preparation method according to claim 1, wherein In step S34, the volume ratio of acetonitrile to water in the high performance liquid chromatography mobile phase is 17:83; and the volume proportion of trifluoroacetic acid in the mobile phase is 0.1%.

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