Diterpenoid alkaloid compound in delphinium lanceolata as well as preparation method and application of diterpenoid alkaloid compound

Abstract

The invention relates to a method for separating a diterpenoid alkaloid compound from delphinium lanceolata and application of the diterpenoid alkaloid compound. According to the compound, delphinium lanceolata is taken as a raw material, solvent extraction, acid dissolution and alkali precipitation treatment and solvent extraction are performed, and six new diterpenoid alkaloid monomeric compounds are obtained by comprehensively utilizing normal-phase silica gel column chromatography, reverse-phase silica gel column chromatography, reverse-phase column chromatography, sephadex LH-20 and semi-preparative high performance liquid chromatography. The obtained compound is subjected to analgesic activity determination, and experimental results show that the diterpenoid alkaloid compound separated from delphinium lanceolata has analgesic activity and can be used for preparing analgesic drugs.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a diterpene alkaloid compound from Delphinium tricholoma flower, a preparation method and a use thereof. Background Art

[0002] Diterpenoid alkaloids are heterocyclic systems containing β-aminoethanol, methylamine or ethylamine nitrogen atoms formed by the amination of tetracyclic or pentacyclic diterpenes. They are mainly found in plants of the genera Aconitum and Delphinium in the Ranunculaceae family and have significant analgesic, antiarrhythmic, anti-inflammatory and anti-tumor activities.

[0003] Delphinium is a genus in the Ranunculaceae family, primarily distributed in northern temperate regions, with over 300 species worldwide. In China, Delphinium species are extremely diverse, found almost throughout the country. The lasiocarpum (Delphinium naviculare var. WTWang), a dicotyledonous plant and a variant of the lasiocarpum (Delphinium naviculare WTWang), is native to the western Tianshan Mountains and the western Junggar Mountains, from Gongliu to Tuoli in Xinjiang, and is endemic to Xinjiang.

[0004] The analgesic activity of diterpenoid alkaloids has been extensively studied in China. Currently, 3-acetylaconitine, lappaconitine, and kusalaconitine have been developed as non-addictive analgesics for clinical use. Diterpenoid alkaloids are both the primary pharmacological components of Aconitum and Delphinium plants and the primary source of their toxicity. Therefore, identifying lead compounds with excellent analgesic efficacy and low toxicity is a primary research goal. Currently, the extraction, isolation, structural identification, and related pharmacological studies of diterpenoid alkaloids from the flowers of Delphinium tricholoma are still in their early stages. Therefore, systematic and in-depth research on the diterpenoid alkaloid components of Delphinium tricholoma is of great significance to clarify the material basis of their analgesic activity and to discover new, specifically active diterpenoid alkaloid compounds and highly effective, low-toxicity drug leads. Summary of the Invention

[0005] The present invention aims to provide diterpenoid alkaloid compounds from the flowers of Delphinium tricholoma. The compounds are extracted from the medicinal material of Delphinium tricholoma by solvent extraction, followed by acid-base treatment and solvent extraction. Separation is performed by two, three, or four of the following methods: normal-phase silica gel column chromatography, reversed-phase silica gel (ODS) column chromatography, reversed-phase (MCI) column chromatography, Sephadex LH-20, or semi-preparative high-performance liquid chromatography. Six new diterpenoid alkaloids are obtained: Compound 1, delphinium tricholoma A; Compound 2, delphinium tricholoma B; Compound 3, delphinium tricholoma D; Compound 4, delphinium tricholoma G; Compound 5, delphinidine A; and Compound 6, delphinidine B. The obtained compounds were tested for analgesic activity, and the results showed that compounds delphinium A, B, D, G, and I exhibited moderate analgesic activity in an acetic acid-induced writhing mouse model and can be used to prepare analgesic drugs.

[0006] The diterpene alkaloid compound isolated from the flower of Delphinium tricholoma described in the present invention has the structural formula:

[0007]

[0008] in:

[0009] Compound 1 is named Delphinidinium tricholoma A;

[0010] Compound 2 is named Delphinium tricholoma B;

[0011] Compound 3 is named Delphinium tricholoma D;

[0012] Compound 4 is named Delphinidinium tricholoma G;

[0013] Compound 5 is named delphinidin A;

[0014] Compound 6 is named delphinidin B;

[0015] Compounds 5 and 6 are a pair of regioisomeric mixtures.

[0016] The method for isolating diterpenoid alkaloid compounds from Delphinium tricholoma flowers is carried out according to the following steps:

[0017] a. Grind the whole plant of Delphinium tricholoma, extract with 10-95% ethanol aqueous solution, methanol or dichloromethane at room temperature by cold soaking, percolation, heating under reflux or ultrasonic extraction, and concentrate under reduced pressure to recover the solvent to obtain an extract;

[0018] b. Dispersing the extract obtained in step a with 1-5% by mass sulfuric acid or hydrochloric acid, adjusting the pH of the obtained acidic aqueous layer to 9-12 with anhydrous sodium carbonate, aqueous ammonia or sodium hydroxide, and then extracting with organic solvents such as chloroform, ethyl acetate or n-butanol, and concentrating under reduced pressure to recover the organic solvent to obtain total alkaloids;

[0019] c. Separating the total alkaloids obtained in step b by two, three or four methods selected from the group consisting of normal phase silica gel column chromatography, reverse phase silica gel column chromatography, reverse phase column chromatography, Sephadex LH-20 or semi-preparative high performance liquid chromatography;

[0020] d. Detect and analyze the product using chromatography or high performance liquid chromatography to obtain new diterpenoid alkaloid compounds.

[0021] Two separation methods in step c:

[0022] Using a combination of normal phase silica gel column chromatography and semi-preparative high performance liquid chromatography, or using a combination of reverse phase silica gel column chromatography and semi-preparative high performance liquid chromatography:

[0023] The total alkaloid extract obtained was separated by silica gel column chromatography using gradient elution with dichloromethane and methanol in a volume ratio of 100:1, 50:1, 20:1, 10:1, and 1:1 or methanol-water in a volume ratio of 10:90-100:0. The fractions were analyzed by silica gel thin layer chromatography, and the same fractions were combined to obtain 9 components AI;

[0024] The obtained fraction D was further separated by normal-phase silica gel column chromatography or reverse-phase silica gel column chromatography using a gradient elution with dichloromethane and methanol in a volume ratio of 100:1, 50:1, 20:1, 10:1, and 1:1 or methanol-water in a volume ratio of 10:90-100:0. After TLC analysis, the same fractions were combined to obtain compound delphinidinium tricholoma B and a secondary fraction Fr.D10.

[0025] Fr.D10 was separated by normal phase silica gel column chromatography or reverse phase silica gel column chromatography, with isocratic elution using petroleum ether-acetone in a volume ratio of 5:1 or gradient elution using methanol-water in a volume ratio of 40:60-100:0. After TLC analysis, the same fractions were combined to obtain Fr.D10e. Fr.D10e was purified by semi-preparative high performance liquid chromatography, with isocratic elution using acetonitrile-water in a volume ratio of 28:72 and 0.1% formic acid to obtain compound delphinidinium tricholometasum G.

[0026] The obtained fraction E was separated by normal-phase silica gel column chromatography or reverse-phase silica gel column chromatography, using a gradient elution method with petroleum ether-acetone in a volume ratio of 10:1, 5:1, or 3:1, or methanol-water in a volume ratio of 30:70-100:0. The obtained fractions were analyzed by TLC and then combined to obtain the Fr.E42 fraction. Fr.E42 was purified by semi-preparative high-performance liquid chromatography, using acetonitrile-water in a volume ratio of 24:76 and 0.1% formic acid as the isocratic elution method to obtain the compounds delphinidin A / B, which are a mixture of a pair of regioisomers.

[0027] The obtained F fraction was separated by normal phase silica gel column chromatography or reverse phase silica gel column chromatography, and gradient eluted with chloroform-methanol with a volume ratio of 70:1, 60:1, 50:1, 20:1, 9:1, 8:2, 7:3, 6:4, 5:5 or methanol-water with a volume ratio of 10:90-100:0. The obtained fractions were detected by TLC and the same parts were combined to obtain Fr.F1-Fr.F11 fractions; the Fr.F4 fraction was separated by normal phase silica gel column chromatography. Separate the product by column chromatography or reverse-phase silica gel column chromatography, using gradient elution with chloroform-methanol in volume ratios of 25:1, 20:1, and 15:1 or methanol-water in volume ratios of 40:60-100:0. The resulting fractions are analyzed by TLC and then combined to obtain fraction Fr.F4a. Fr.F4a is purified by semi-preparative high-performance liquid chromatography, using isocratic elution with acetonitrile-water in a volume ratio of 35:65 and 0.1% formic acid to obtain a new compound, delphinidinium tricholometasum D.

[0028] The obtained fraction I was separated and purified by normal phase silica gel column chromatography or reverse phase silica gel column chromatography, and gradient eluted with chloroform-methanol with a volume ratio of 50:1, 20:1, 9:1, 8:2, 7:3, 6:4, 5:5 or methanol-water with a volume ratio of 10:90-100:0. After TLC detection, the same fractions were combined to obtain the secondary fraction Fr.I1; Fr.I1 was further separated by normal phase silica gel column chromatography or reverse phase silica gel column chromatography. Purification was carried out by gradient elution with chloroform-methanol in a volume ratio of 20:1, 9:1, 8:2, 7:3, 6:4, and 5:5 or methanol-water in a volume ratio of 40:60-100:0. After TLC detection, the same fractions were combined to obtain Fr.I4a. Fr.I4a was purified by semi-preparative high-performance liquid chromatography and isocratically eluted with acetonitrile-water in a volume ratio of 23:77 and 0.1% formic acid to obtain a new compound, Delphinidinium tricholometaphyllum A.

[0029] Three separation methods in step c:

[0030] Using a combination of normal phase silica gel column chromatography, reverse phase silica gel column chromatography and semi-preparative high performance liquid chromatography, or using a combination of reverse phase silica gel column chromatography, normal phase silica gel column chromatography and semi-preparative high performance liquid chromatography:

[0031] The obtained total alkaloid extract was separated by normal phase silica gel column chromatography or reverse phase silica gel column chromatography, using a gradient elution with dichloromethane and methanol in a volume ratio of 100:1, 50:1, 20:1, 10:1, and 1:1 or methanol-water in a volume ratio of 10:90-100:0 as the eluent. The fractions were analyzed by TLC, and the same fractions were combined to obtain 9-11 components AI;

[0032] The obtained fraction D was separated and purified by reverse phase silica gel column chromatography or normal phase silica gel column chromatography, using a gradient elution with methanol-water in a volume ratio of 10:90-100:0 or sequentially with dichloromethane and methanol in a volume ratio of 100:1, 50:1, 20:1, 10:1, and 1:1. After TLC detection, the same fractions were combined to obtain a new compound delphinidin B and a secondary fraction Fr.D10. Fr.D10 was separated by normal phase silica gel column chromatography or reverse phase silica gel column chromatography, using isocratic elution with petroleum ether-acetone in a volume ratio of 5:1 or methanol-water in a volume ratio of 10:90-100:0. After TLC detection, the same fractions were combined to obtain a fraction Fr.D10e. Fr.D10e was purified by semi-preparative high performance liquid chromatography using isocratic elution with acetonitrile-water in a volume ratio of 28:72 and 0.1% formic acid to obtain a new compound delphinidin G.

[0033] The obtained E was separated by reverse phase silica gel column chromatography or normal phase silica gel column chromatography, sequentially eluting with a gradient of methanol-water in a volume ratio of 30:70-100:0 or petroleum ether-acetone in a volume ratio of 10:1, 5:1, and 3:1. The obtained fractions were analyzed by TLC and then combined to obtain fraction Fr.E42. The Fr.E42 fraction was purified by semi-preparative high performance liquid chromatography, isocratically eluting with acetonitrile-water in a volume ratio of 24:76 and 0.1% formic acid to obtain compounds delphinidin A / B, which are a mixture of a pair of regioisomers.

[0034] The obtained F fraction was separated by reverse phase silica gel column chromatography or normal phase silica gel column chromatography, and gradient eluted with methanol-water in a volume ratio of 10:90-100:0 or chloroform-methanol in a volume ratio of 70:1, 60:1, 50:1, 20:1, 9:1, 8:2, 7:3, 6:4, 5:5. The obtained fractions were detected by TLC and the same parts were combined to obtain fraction Fr.F4. The Fr.F4 fraction was separated by normal phase silica gel column chromatography. The product is separated by chromatography or reverse-phase silica gel column chromatography using a gradient elution with chloroform-methanol in a volume ratio of 25:1, 20:1, or 15:1 or methanol-water in a volume ratio of 40:60-100:0. The resulting fractions are analyzed by TLC and then combined to obtain fraction Fr.F4a. Fr.F4a is purified by semi-preparative high-performance liquid chromatography using an isocratic elution with acetonitrile-water in a volume ratio of 35:65 and 0.1% formic acid to obtain a new compound, Delphinidinium tricholometaphyllum D.

[0035] The obtained fraction I was separated by reverse phase silica gel column chromatography or normal phase silica gel column chromatography, and gradient eluted with methanol-water in a volume ratio of 10:90-100:0 or chloroform-methanol in a volume ratio of 50:1, 20:1, 9:1, 8:2, 7:3, 6:4, 5:5. After TLC detection, the same fractions were combined to obtain a secondary fraction Fr.I4; Fr.I4 was separated by normal phase silica gel column chromatography or reverse phase silica gel column chromatography. Purification was performed by gradient elution using chloroform-methanol with a volume ratio of 20:1, 9:1, 8:2, 7:3, 6:4, and 5:5 or methanol-water with a volume ratio of 40:60-100:0. After TLC detection, the same fractions were combined to obtain Fr.I4a. Fr.I4a was purified by semi-preparative high-performance liquid chromatography using acetonitrile-water with a volume ratio of 23:77 and 0.1% formic acid as the isocratic eluent to obtain a new compound, Delphinidinium tricholometaphyllum A.

[0036] Four separation methods in step c:

[0037] Using a combination of normal phase silica gel column chromatography, reverse phase silica gel column chromatography, Sephadex LH-20 and semi-preparative high performance liquid chromatography, or using a combination of reverse phase silica gel column chromatography, reverse phase column, Sephadex LH-20 and semi-preparative high performance liquid chromatography:

[0038] The total alkaloid extract obtained was separated by normal phase silica gel column chromatography or reverse phase silica gel column chromatography, and gradient eluted with dichloromethane and methanol in a volume ratio of 100:1, 50:1, 20:1, 10:1, and 1:1, or with methanol-water in a volume ratio of 10:90-100:0. The fractions were analyzed by normal phase silica gel thin layer chromatography, and the same fractions were combined to obtain 9 components AI;

[0039] The obtained fraction D was separated and purified using a reverse phase column or Sephadex LH-20, and gradient or isocratic elution was performed with methanol-water in a volume ratio of 10:90-100:0 or dichloromethane-methanol in a volume ratio of 1:1. After TLC detection, the same fractions were combined to obtain a new compound delphinidin B and a secondary fraction Fr.D10. Fr.D10 was separated and purified using Sephadex LH-20 column chromatography or a reverse phase column, and isocratic or gradient elution was performed with dichloromethane-methanol in a volume ratio of 1:1 or methanol-water in a volume ratio of 40:60-100:0. After TLC detection, the same fractions were combined to obtain a fraction Fr.D10e. Fr.D10e was purified by semi-preparative high performance liquid chromatography, and isocratic elution was performed with acetonitrile-water in a volume ratio of 28:72, 0.1% formic acid to obtain a new compound delphinidin G.

[0040] The obtained fraction E is separated using Sephadex LH-20 or a reverse phase column and isocratically or gradiently eluted with dichloromethane-methanol in a volume ratio of 1:1 or methanol-water in a volume ratio of 30:70-100:0. The obtained fractions are analyzed by TLC and then combined to obtain fraction Fr.E42. Fr.E42 is purified by semi-preparative high performance liquid chromatography and isocratically eluted with acetonitrile-water in a volume ratio of 24:76 and 0.1% formic acid to obtain compounds delphinidin A / B, which are a mixture of a pair of regioisomers.

[0041] The obtained F fraction was separated by normal phase silica gel column chromatography or reverse phase silica gel column chromatography, and gradient eluted with chloroform-methanol with a volume ratio of 70:1, 60:1, 50:1, 20:1, 9:1, 8:2, 7:3, 6:4, 5:5 or methanol-water with a volume ratio of 10:90-100:0. The obtained fractions were detected by TLC and the same parts were combined to obtain the Fr.F4 fraction; the Fr.F4 fraction was separated by elution. Separate using Sephadex LH-20 or reverse-phase column chromatography, isocratically eluting with a 1:1 volume ratio of dichloromethane-methanol or a 40:60-100:0 volume ratio of methanol-water. After TLC analysis, combine the same fractions to obtain fraction Fr.F4a. Fr.F4a is purified by semi-preparative HPLC, isocratically eluting with a 35:65 volume ratio of acetonitrile-water, 0.1% formic acid, to obtain a new compound, delphinidinium tricholometasum D.

[0042] The obtained fraction I was separated and purified using a reverse phase column or Sephadex LH-20, and gradient or isocratic elution was performed with methanol-water or pure methanol in a volume ratio of 40:60-100:0. After TLC detection, the same fractions were combined to obtain a secondary fraction Fr.I4; Fr.I4 was separated and purified using Sephadex LH-20 or a reverse phase column, and isocratic or gradient elution was performed with methanol or methanol-water in a volume ratio of 40:60-100:0. After TLC detection, the same fractions were combined to obtain a fraction Fr.I4a; Fr.I4a was purified by semi-preparative high performance liquid chromatography, and isocratic elution was performed with acetonitrile-water in a volume ratio of 23:77 and 0.1% formic acid to obtain a new compound delphinidinium tricholometaphyllum A.

[0043] The invention relates to a use of the diterpenoid alkaloid compound isolated from the flower of Delphinium tricholoma in preparing analgesic drugs.

[0044] The present invention discloses a diterpene alkaloid compound from the flower of Delphinium tricholoma, a preparation method and a use thereof. The compound 1 is named Delphinium tricholoma A, which is a white amorphous powder. The results of HRESI(+)MS (m / z 744.3583[M+H] + , theoretical value 744.3590) to determine its molecular formula is C 39 H 53 NO 13 ;according to 1 H. 13 C and 2D NMR data confirmed its structure and named it as delphinium tricholoma A. 1 H and 13 C NMR data assignments are shown in Table 1 [600 MHz ( 1 H), 150MHz( 13 C), solvent: CD3OD];

[0045] Compound 2 is delphinium tricholoma B, which is white needle-shaped crystals. HRESI(+)MS (m / z 436.2684[M+H] + , theoretical value 436.2694) to determine its molecular formula is C 24 H 37 NO6; According to 1 H. 13 C and two-dimensional nuclear magnetic resonance data and X-ray single crystal diffraction to determine its structure, and named it as delphinium tricholoma B. 1 H and 13 C NMR data assignments are shown in Table 1 [600 MHz ( 1 H), 150MHz( 13 C), solvent: CDCl3];

[0046] Compound 3 is delphinium tricholoma D, which is white needle-shaped crystals. HRESI(+)MS (m / z 422.2525[M+H] + , theoretical value 422.2537) to determine its molecular formula is C 23 H 35 NO6; According to 1 H. 13 Its structure was confirmed by C, two-dimensional nuclear magnetic resonance data and X-ray single crystal diffraction, and it was named as Delphinium tricholoma D. 1 H and 13 C NMR data assignments are shown in Table 1 [600 MHz ( 1 H), 150MHz( 13 C), solvent: CDCl3];

[0047] Compound 4 is named as Delphinidinium tricholoma G, which is a white amorphous powder. It was characterized by HRESI(+)MS (m / z 697.3320[M+H] + , theoretical value 697.3331) to determine its molecular formula is C 37 H 48 N2O 11 ;according to 1 H. 13 C and 2D NMR data confirmed its structure and named it as delphinium tricholoma G. 1 H and 13 C NMR data assignments are shown in Table 1 [600 MHz ( 1 H), 150MHz( 13 C), solvent: CD3OD];

[0048] Compound 5 is named as delphinidin A, and compound 6 is named as delphinidin B. Compounds 5 and 6 are a pair of regioisomers, which are white amorphous powders. + , theoretical value 729.3593) to determine its molecular formula is C 38 H 52 N2O 11 ;according to 1 H. 13 C and 2D NMR data confirmed their structures, and they were named delphinidin A / B, respectively. 1 H and 13 C NMR data assignments are shown in Table 1 [600 MHz ( 1 H), 150MHz( 13 C), solvent: CD3OD].

[0049] Table 1 Compounds 1-3 1 H (600MHz) and13 C (150 MHz) NMR data [δ (ppm), J (Hz)]

[0050]

[0051]

[0052] Table 2 Compounds 3-6 1 H (600MHz) and 13 C (150 MHz) NMR data [δ (ppm), J (Hz)]

[0053]

[0054] BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 The compound of the present invention 1 is the 1 H NMR spectrum;

[0056] Figure 2 The compound 1 of the present invention is the 13 C NMR spectrum;

[0057] Figure 3 The compound 2 of the present invention is the 1 H NMR spectrum;

[0058] Figure 4 The compound 2 of the present invention is the 13 C NMR spectrum;

[0059] Figure 5 The compound 3-delphinidin D of the present invention 1 H NMR spectrum;

[0060] Figure 6 The compound 3-delphinidin D of the present invention 13 C NMR spectrum;

[0061] Figure 7 The compound of the present invention 4-delphinidin G 1 H NMR spectrum;

[0062] Figure 8 The compound of the present invention 4-delphinidin G 13 C NMR spectrum;

[0063] Figure 9 The compound 5-delphinidin A / B of the present invention 1 H NMR spectrum;

[0064] Figure 10 The compound 5-delphinidin A / B of the present invention 13 C NMR spectrum. DETAILED DESCRIPTION

[0065] The present invention discloses a diterpene alkaloid compound from the flower of Delphinium tricholoma, as well as its preparation method and use. All reagents used are analytical grade, and the acetonitrile used in the high-performance liquid chromatography (HPLC) is HPLC grade (Merk, USA). Column chromatography silica gel (100-200 mesh, 200-300 mesh): produced by Qingdao Ocean Chemical Plant; Sephadex LH-20 gel: produced by Pharmacia, Sweden. High-performance liquid chromatography (Agilent, USA): P680 HPLC pump, ASI-100 autosampler, TCC-100 column oven, UVD170U UV detector (quad wavelength), quaternary eluent, online degasser, Chromeleon chromatography workstation. Preparative high-performance liquid chromatography (Jiangsu Hanbang Technology Co., Ltd.): NP7005C pump, N3000D UV detector (dual wavelength), semi-preparative dynamic mixer, EasyChrom-1000 chromatography workstation. Mass spectrometry was performed using an ultra-high performance liquid chromatography-tandem quadrupole electrostatic field orbitrap mass spectrometer: UHPLC-Q-Exactive Orbitrap MS (Thermo Fisher, MA, USA); nuclear magnetic resonance was performed using a Bruker AVANCE NEO 600 nuclear magnetic resonance instrument (Bruker, USA); an electronic balance (METTER AC-100, Sartorious, Germany); a rotary evaporator (N-1001D, Shanghai Ailang Instrument Co., Ltd.); and a high-precision polarimeter (UAutopol VI, Rudolph, USA).

[0066] The flower of Delphinium naviculare var. lasiocarpum was collected in Gongliu County, Ili Kazakh Autonomous Prefecture, Xinjiang Uygur Autonomous Region, China in August 2022. It was identified as Delphinium naviculare var. lasiocarpum W.T. Wang, a plant of the genus Delphinium in the Ranunculaceae family (Ranunculeceae), by Researcher Liu Geyu of the Xinjiang Institute of Physics and Chemistry, Chinese Academy of Sciences. The specimen is preserved in the Xinjiang Institute of Physics and Chemistry, Chinese Academy of Sciences.

[0067] Example 1 (two separations)

[0068] a. Grind 15.0 kg of the whole herb of Delphinium pubescens and extract it with 80 L of dichloromethane solution by percolation at room temperature. Evaporate the solvent under reduced pressure to obtain a crude extract of Delphinium pubescens;

[0069] b. Dispersing the crude extract obtained in step a with a 1-5% by mass sulfuric acid solution, adjusting the pH of the obtained acidic aqueous layer to 10 with Na2CO3, extracting with dichloromethane, and evaporating the dichloromethane under reduced pressure to obtain a total alkaloid extract;

[0070] c. Separate the obtained total alkaloid extract by normal phase silica gel column chromatography using a gradient elution method with dichloromethane and methanol in a volume ratio of 100:1, 50:1, 20:1, 10:1, and 1:1 as eluents. The fractions were analyzed by silica gel thin layer chromatography (TLC). The same fractions were combined to obtain 9 components AI;

[0071] The obtained fraction D was further separated by normal phase silica gel column chromatography using a gradient elution with dichloromethane and methanol in a volume ratio of 100:1, 50:1, 20:1, 10:1, and 1:1 as eluents. After TLC analysis, the same fractions were combined to obtain a new compound delphinidin B and a secondary fraction Fr.D10. Fr.D10 was separated by normal phase silica gel column chromatography using isocratic elution with petroleum ether-acetone in a volume ratio of 5:1. After TLC analysis, the same fractions were combined to obtain a fraction Fr.D10e. Fr.D10e was purified by semi-preparative high performance liquid chromatography using isocratic elution with acetonitrile-water in a volume ratio of 28:72 and 0.1% formic acid to obtain a new compound delphinidin G.

[0072] The obtained fraction E was separated by normal phase silica gel column chromatography, and gradient elution was performed with petroleum ether-acetone in a volume ratio of 10:1, 5:1, and 3:1, respectively. The obtained fractions were analyzed by TLC and the same fractions were combined to obtain the Fr.E42 fraction. Fr.E42 was purified by semi-preparative high performance liquid chromatography, and isocratically eluted with acetonitrile-water in a volume ratio of 24:76 and 0.1% formic acid to obtain the compound delphinidin A / B, which is a mixture of a pair of regioisomers.

[0073] The obtained fraction F was separated by normal phase silica gel column chromatography and gradient eluted with chloroform-methanol in a volume ratio of 70:1, 60:1, 50:1, 20:1, 9:1, 8:2, 7:3, 6:4, and 5:5, respectively. The obtained fractions were analyzed by TLC and the same fractions were combined to obtain fractions Fr.F1-Fr.F11. The Fr.F4 fraction was separated by normal phase silica gel column chromatography and gradient eluted with chloroform-methanol in a volume ratio of 25:1, 20:1, and 15:1, respectively. The obtained fractions were analyzed by TLC and the same fractions were combined to obtain fraction Fr.F4a. Fr.F4a was purified by semi-preparative high performance liquid chromatography and isocratically eluted with acetonitrile-water in a volume ratio of 35:65 and 0.1% formic acid to obtain a new compound, Delphinidinium tricholometaphyllum D.

[0074] The obtained fraction I was separated and purified using a normal phase silica gel column, and gradient eluted with chloroform-methanol in a volume ratio of 50:1, 20:1, 9:1, 8:2, 7:3, 6:4, and 5:5, respectively. After TLC detection, the same fractions were combined to obtain a second fraction Fr.I1; Fr.I1 was further separated and purified using a normal phase silica gel column, and gradient eluted with chloroform-methanol in a volume ratio of 20:1, 9:1, 8:2, 7:3, 6:4, and 5:5, respectively. After TLC detection, the same fractions were combined to obtain a fraction Fr.I4a; Fr.I4a was purified by semi-preparative high performance liquid chromatography, and eluted isocratically with acetonitrile-water in a volume ratio of 23:77 and 0.1% formic acid to obtain a new compound delphinidinium pubescens A.

[0075] Example 2 (two separations)

[0076] a. Grind 15.0 kg of the whole herb of Delphinium tricholoma and extract it by percolation with 80 L of 80% ethanol at room temperature. Evaporate the solvent under reduced pressure to obtain a crude extract of Delphinium tricholoma;

[0077] b. Dispersing the crude extract obtained in step a with 1-5% by mass of sulfuric acid, adjusting the pH of the obtained acidic aqueous layer to 10 with aqueous ammonia, extracting with dichloromethane, and evaporating the dichloromethane under reduced pressure to obtain a total alkaloid extract;

[0078] c. Separate the obtained total alkaloid extract by reverse phase silica gel (ODS) column chromatography, using a gradient elution of methanol-water in a volume ratio of 10:90 to 100:0. The fractions were analyzed by TLC, and the same fractions were combined to obtain 9 components AI;

[0079] The obtained fraction D was separated and purified by reverse-phase silica gel (ODS) column chromatography, using a gradient elution of methanol-water in a volume ratio of 10:90-100:0. After TLC analysis, the same fractions were combined to obtain a new compound delphinidin B and a secondary fraction Fr.D10. Fr.D10 was separated and purified by reverse-phase silica gel (ODS) column chromatography, using a gradient elution of methanol-water in a volume ratio of 40:60-100:0. After TLC analysis, the same fractions were combined to obtain a fraction Fr.D10e. Fr.D10e was purified by semi-preparative high-performance liquid chromatography, using an isocratic elution of acetonitrile-water in a volume ratio of 28:72, 0.1% formic acid to obtain a new compound delphinidin G.

[0080] The resulting fraction E was separated by reverse-phase silica gel (ODS) column chromatography using a gradient elution with methanol-water in a ratio of 30:70 to 100:0, by volume. The resulting fractions were analyzed by TLC and combined to obtain fraction Fr.E42. Fr.E42 was purified by semi-preparative high-performance liquid chromatography using acetonitrile-water in a ratio of 24:76, by volume, and isocratic elution with 0.1% formic acid to obtain compounds delphinidin A / B, which are a mixture of a pair of regioisomers.

[0081] The resulting fraction F was subjected to reverse-phase silica gel (ODS) column chromatography using a gradient elution of methanol-water (volume ratio) of 10:90-100:0. The resulting fractions were analyzed by TLC and combined to obtain fraction Fr.F4. The Fr.F4 fraction was further separated by reverse-phase silica gel (ODS) column chromatography using a gradient elution of methanol-water (volume ratio) of 40:60-100:0. The resulting fractions were analyzed by TLC and combined to obtain fraction Fr.F4a. Fr.F4a was purified by semi-preparative high-performance liquid chromatography using isocratic elution with acetonitrile-water (volume ratio) of 35:65 and 0.1% formic acid to obtain a new compound, Delphinidinium tricholoma D.

[0082] The obtained fraction I was subjected to reverse-phase silica gel (ODS) column chromatography, and gradient elution was performed with methanol-water in a volume ratio of 10:90-100:0. After TLC detection, the same fractions were combined to obtain the secondary fraction Fr.I4; Fr.I4 was further subjected to reverse-phase silica gel (ODS) column chromatography, and gradient elution was performed with methanol-water in a volume ratio of 40:60-100:0. After TLC detection, the same fractions were combined to obtain the fraction Fr.I4a; Fr.I4a was purified by semi-preparative high-performance liquid chromatography, and the new compound delphinidinium tricholoma A was obtained by isocratic elution with acetonitrile-water in a volume ratio of 23:77 and 0.1% formic acid.

[0083] Example 3 (three separations)

[0084] a. Grind 15.0 kg of the whole herb of Delphinium tricholoma, extract it with 80 L of methanol by percolation, and concentrate under reduced pressure to recover methanol to obtain a total extract;

[0085] b. Dispersing the total extract obtained in step a in a 1-3% by mass aqueous solution of hydrochloric acid, filtering, adjusting the pH of the obtained aqueous solution to 10 with Na2CO3, extracting with ethyl acetate, and evaporating the ethyl acetate under reduced pressure to obtain a total alkaloid extract;

[0086] c. Separate the obtained total alkaloid extract by normal phase silica gel column chromatography using a gradient elution method with dichloromethane and methanol in a volume ratio of 100:1, 50:1, 20:1, 10:1, and 1:1 as eluents. The fractions were analyzed by TLC and the same fractions were combined to obtain 11 components AI;

[0087] The obtained fraction D was separated and purified by reverse phase silica gel (ODS) column chromatography, using a gradient elution with methanol-water in a volume ratio of 10:90-100:0. After TLC analysis, the same fractions were combined to obtain a new compound delphinidin B and a secondary fraction Fr.D10. Fr.D10 was separated by normal phase silica gel column chromatography, using isocratic elution with petroleum ether-acetone in a volume ratio of 5:1. After TLC analysis, the same fractions were combined to obtain a fraction Fr.D10e. Fr.D10e was purified by semi-preparative high performance liquid chromatography, using isocratic elution with acetonitrile-water in a volume ratio of 28:72, 0.1% formic acid to obtain a new compound delphinidin G.

[0088] The obtained E was separated by reverse-phase silica gel (ODS) column chromatography using a gradient elution with methanol-water in a volume ratio of 30:70 to 100:0. The obtained fractions were analyzed by TLC and then combined to obtain fraction Fr.E42. Fr.E42 was purified by semi-preparative high-performance liquid chromatography using acetonitrile-water in a volume ratio of 24:76 and 0.1% formic acid to obtain compounds delphinidin A / B, which are a mixture of a pair of regioisomers.

[0089] The resulting fraction F was separated by reverse-phase silica gel (ODS) column chromatography, using a gradient elution of methanol-water (volume ratio) of 10:90-100:0. The resulting fractions were analyzed by TLC and combined to obtain fraction Fr.F4. The Fr.F4 fraction was separated by normal-phase silica gel column chromatography, using a gradient elution of chloroform-methanol (volume ratio) of 25:1, 20:1, and 15:1, respectively. The resulting fractions were analyzed by TLC and combined to obtain fraction Fr.F4a. Fr.F4a was purified by semi-preparative high-performance liquid chromatography, using an isocratic elution of acetonitrile-water (volume ratio) of 35:65 and 0.1% formic acid to obtain a new compound, Delphinidinium tricholometaphyllum D.

[0090] The obtained fraction I was separated by reverse phase silica gel (ODS) column chromatography, and gradient elution was performed with methanol-water in a volume ratio of 10:90-100:0. After TLC detection, the same fractions were combined to obtain the second fraction Fr.I4; Fr.I4 was separated and purified by normal phase silica gel column, and gradient elution was performed with chloroform-methanol in a volume ratio of 20:1, 9:1, 8:2, 7:3, 6:4, and 5:5, respectively. After TLC detection, the same fractions were combined to obtain the fraction Fr.I4a; Fr.I4a was purified by semi-preparative high performance liquid chromatography, and isocratic elution was performed with acetonitrile-water in a volume ratio of 23:77 and 0.1% formic acid to obtain the new compound delphinidinium tricholometaphyllum A.

[0091] Example 4 (three separations)

[0092] a. Grind 15.0 kg of the whole herb of Delphinium tricholoma and extract it by percolation with 75 L of dichloromethane and methanol at a volume ratio of 1:1 at room temperature. Evaporate the solvent under reduced pressure to obtain a crude extract of Delphinium tricholoma;

[0093] b. Dispersing the crude extract obtained in step a with 1-5% by mass hydrochloric acid, adjusting the pH of the obtained acid solution to 10 with aqueous ammonia, extracting with ethyl acetate, and evaporating the ethyl acetate under reduced pressure to obtain a total alkaloid extract;

[0094] c. Separate the obtained total alkaloid extract by reverse phase silica gel (ODS) column chromatography, using a gradient elution with methanol-water in a volume ratio of 10:90-100:0. The fractions were analyzed by silica gel thin layer chromatography (TLC), and the same fractions were combined to obtain 9 components AI;

[0095] The obtained fraction D was further separated by normal phase silica gel column chromatography using gradient elution with dichloromethane and methanol in a volume ratio of 100:1, 50:1, 20:1, 10:1, and 1:1, respectively. After TLC analysis, the same fractions were combined to obtain a new compound delphinidin B and a secondary fraction Fr.D10. Fr.D10 was separated by reverse phase silica gel (ODS) column chromatography using a gradient elution with methanol-water in a volume ratio of 10:90-100:0. After TLC analysis, the same fractions were combined to obtain a fraction Fr.D10e. Fr.D10e was purified by semi-preparative high performance liquid chromatography using isocratic elution with acetonitrile-water in a volume ratio of 28:72 and 0.1% formic acid to obtain a new compound delphinidin G.

[0096] The obtained fraction E was separated by normal phase silica gel column chromatography and gradient eluted with petroleum ether-acetone in a volume ratio of 10:1, 5:1, and 3:1, respectively. The obtained fractions were analyzed by TLC and the same fractions were combined to obtain fraction Fr.E42. Fr.E42 was purified by semi-preparative high performance liquid chromatography using acetonitrile-water in a volume ratio of 24:76 and 0.1% formic acid to obtain compound delphinidin A / B, which is a mixture of a pair of regioisomers.

[0097] The resulting fraction F was subjected to normal phase silica gel column chromatography, sequentially eluting with chloroform-methanol in a volume ratio of 70:1, 60:1, 50:1, 20:1, 9:1, 8:2, 7:3, 6:4, and 5:5. The resulting fractions were analyzed by TLC and combined to obtain fraction Fr.F4. The Fr.F4 fraction was separated by reverse phase silica gel (ODS) column chromatography, gradient eluting with methanol-water in a volume ratio of 40:60 to 100:0. The resulting fractions were analyzed by TLC and combined to obtain fraction Fr.F4a. Fr.F4a was purified by semi-preparative high performance liquid chromatography, isocratically eluting with acetonitrile-water in a volume ratio of 35:65 and 0.1% formic acid to obtain a new compound, Delphinidinium tricholometaphyllum D.

[0098] The obtained fraction I was separated and purified using a normal phase silica gel column, and gradient eluted with chloroform-methanol in a volume ratio of 50:1, 20:1, 9:1, 8:2, 7:3, 6:4, and 5:5, respectively. After TLC detection, the same fractions were combined to obtain a secondary fraction Fr.I4; Fr.I4 was further separated using reverse phase silica gel (ODS) column chromatography, and gradient eluted with methanol-water in a volume ratio of 40:60-100:0, and the same fractions were combined after TLC detection to obtain a fraction Fr.I4a; Fr.I4a was purified by semi-preparative high performance liquid chromatography, and eluted isocratically with acetonitrile-water in a volume ratio of 23:77 and 0.1% formic acid to obtain a new compound delphinidinium tricholometaphyllum A.

[0099] Example 5 (four separations)

[0100] a. Grind 15.0 kg of the whole plant of Delphinium tricholoma, extract it with 80 L of 80% ethanol under reflux at room temperature, and evaporate the solvent under reduced pressure to obtain a crude extract of Delphinium tricholoma;

[0101] b. Dispersing the crude extract obtained in step a with 1-3% by mass of sulfuric acid, adjusting the pH of the obtained acid aqueous solution to 10 with 5% NaOH, extracting with dichloromethane, and evaporating under reduced pressure to obtain a total alkaloid extract;

[0102] c. Separate the obtained total alkaloid extract by normal phase silica gel column chromatography using gradient elution with dichloromethane and methanol in a volume ratio of 100:1, 50:1, 20:1, 10:1, and 1:1, respectively. The fractions were analyzed by silica gel thin layer chromatography (TLC). Identical fractions were combined to obtain 9 components AI;

[0103] The obtained fraction D was separated and purified by reverse phase silica gel (ODS) column chromatography, using a gradient elution with methanol-water in a volume ratio of 10:90-100:0. After TLC detection, the same fractions were combined to obtain a new compound delphinidin B and a secondary fraction Fr.D10. Fr.D10 was isocratically eluted with dichloromethane-methanol in a volume ratio of 1:1 using Sephadex LH-20. After TLC detection, the same fractions were combined to obtain a fraction Fr.D10e. Fr.D10e was purified by semi-preparative high performance liquid chromatography, using an isocratic elution with acetonitrile-water in a volume ratio of 28:72, 0.1% formic acid to obtain a new compound delphinidin G.

[0104] The obtained fraction E was separated using Sephadex LH-20 and isocratically eluted with dichloromethane-methanol in a volume ratio of 1:1. The obtained fractions were analyzed by TLC and combined to obtain fraction Fr.E42. Fr.E42 was purified by semi-preparative high performance liquid chromatography using acetonitrile-water in a volume ratio of 24:76 and 0.1% formic acid to obtain compounds delphinidin A / B, which are a mixture of a pair of regioisomers.

[0105] The obtained fraction F was separated using a normal phase silica gel column and gradient eluted with chloroform-methanol in a volume ratio of 70:1, 60:1, 50:1, 20:1, 9:1, 8:2, 7:3, 6:4, and 5:5, respectively. The obtained fractions were analyzed by TLC and the same fractions were combined to obtain fraction Fr.F4. The Fr.F4 fraction was separated using Sephadex LH-20 column chromatography and isocratically eluted with dichloromethane-methanol in a volume ratio of 1:1. The obtained fractions were analyzed by TLC and the same fractions were combined to obtain fraction Fr.F4a. Fr.F4a was purified by semi-preparative high performance liquid chromatography and isocratically eluted with acetonitrile-water in a volume ratio of 35:65 and 0.1% formic acid to obtain a new compound, Delphinidinium tricholometaphyllum D.

[0106] The obtained fraction I was separated using Sephadex LH-20 and isocratically eluted with methanol. After TLC detection, the same parts were combined to obtain fractions Fr.I4a; Fr.I4a was purified by semi-preparative high-performance liquid chromatography and isocratically eluted with acetonitrile-water (volume ratio 23:77) and 0.1% formic acid to obtain a new compound delphinidinium tricholometaphyllum A.

[0107] Example 6 (four separations)

[0108] a. Grind 15.0 kg of the whole herb of Delphinium tricholoma, extract it with 80 L of 75% ethanol aqueous solution by volume at room temperature, and evaporate the solvent under reduced pressure to obtain a crude extract of Delphinium tricholoma;

[0109] b. Dispersing the crude extract obtained in step a with 1-5% by mass sulfuric acid, adjusting the pH value of the obtained acid aqueous solution to 10 with 5% NaOH, and extracting with ethyl acetate to obtain a total alkaloid extract;

[0110] c. Separate the obtained total alkaloid extract by reverse phase silica gel (ODS) column chromatography, using a gradient elution with methanol-water in a volume ratio of 10:90-100:0. The fractions were analyzed by silica gel thin layer chromatography (TLC), and the same fractions were combined to obtain 9 components AI;

[0111] The obtained fraction D was separated and purified using a reverse phase (MCI) column, gradient eluted with methanol-water in a volume ratio of 10:90-100:0. After TLC detection, the same fractions were combined to obtain a new compound delphinidin B and a secondary fraction Fr.D10. Fr.D10 was chromatographed on a Sephadex LH-20 column, isocratically eluted with dichloromethane-methanol in a volume ratio of 1:1. After TLC detection, the same fractions were combined to obtain a fraction Fr.D10e. Fr.D10e was purified by semi-preparative high performance liquid chromatography, isocratically eluted with acetonitrile-water in a volume ratio of 28:72, 0.1% formic acid, to obtain a new compound delphinidin G.

[0112] The obtained fraction E was separated using Sephadex LH-20 and isocratically eluted with dichloromethane-methanol in a volume ratio of 1:1. The obtained fractions were analyzed by TLC and combined to obtain fraction Fr.E42. Fr.E42 was purified by semi-preparative high performance liquid chromatography using acetonitrile-water in a volume ratio of 24:76 and 0.1% formic acid to obtain compounds delphinidin A / B, which are a mixture of a pair of regioisomers.

[0113] The obtained fraction F was eluted with a normal phase silica gel column using a gradient elution method with chloroform-methanol in a volume ratio of 70:1, 60:1, 50:1, 20:1, 9:1, 8:2, 7:3, 6:4, and 5:5, respectively. The obtained fractions were analyzed by TLC and the same fractions were combined to obtain a fraction Fr.F4. The Fr.F4 fraction was isocratically eluted with dichloromethane-methanol in a volume ratio of 1:1 using Sephadex LH-20. The same fractions were combined after TLC analysis to obtain a fraction Fr.F4a. Fr.F4a was purified by semi-preparative high performance liquid chromatography using acetonitrile-water in a volume ratio of 35:65 and 0.1% formic acid to obtain a new compound, delphinidinium tricholometas D.

[0114] The obtained fraction I was separated and purified using a reverse phase (MCI) column, and gradient elution was performed with methanol-water in a volume ratio of 40:60-100:0. After TLC detection, the same fractions were combined to obtain a secondary fraction Fr.I4; Fr.I4 was isocratically eluted with methanol using Sephadex LH-20, and the same fractions were combined after TLC detection to obtain a fraction Fr.I4a; Fr.I4a was purified by semi-preparative high performance liquid chromatography, and isocratically eluted with acetonitrile-water in a volume ratio of 23:77 and 0.1% formic acid to obtain a new compound delphinidinium tricholometaphyllum A.

[0115] Example 7

[0116] The use of the diterpenoid alkaloid compounds isolated from the flowers of Delphinium tricholoma in the present invention in the preparation of analgesic drugs, taking ICR mice as an example:

[0117] Screening of analgesic activity of the obtained diterpenoid alkaloid compounds:

[0118] Experimental animals:

[0119] ICR mice, weighing 18-22 g, half male and half female, were provided by the Department of Experimental Animal Science, Xinjiang Medical University. Mice were housed in an SPF animal experimental center at an indoor temperature of 22 ± 2°C and a relative humidity of 40%-70%, with free access to water and food.

[0120] Experimental instruments and reagents:

[0121] Dimethyl sulfoxide (Adamas), glacial acetic acid (Aladdin), and sodium chloride injection (Xinjiang Huashidan Pharmaceutical Co., Ltd.);

[0122] Experimental content:

[0123] Preparation of drug solution: dissolve the drug in dimethyl sulfoxide or 0.1M / L hydrochloric acid, then dilute with normal saline. The blank control group is the solvent control and stored at room temperature away from light.

[0124] Preparation of 0.6% acetic acid solution: 30 minutes before preparing the model, prepare 10 ml of 10% acetic acid aqueous solution and place it in an ice water box; 25 minutes later, take 10% acetic acid and prepare 0.6% acetic acid solution, place it in an ice water box, and use it immediately after preparation;

[0125] Acetic acid writhing test in mice: 60 ICR mice, half male and half female, were randomly divided into 6 groups: a blank control group and each test drug group, with 10 mice in each group. All mice in each group were intraperitoneally injected with 0.1 mL / 10 g of the drug at a dose of 5 mg / kg. The blank control group was given a blank solvent system. 30 minutes after drug administration, each group of mice was intraperitoneally injected with 0.6% glacial acetic acid (0.1 mL / 10 g). The number of writhing times within 25 minutes was recorded. The pain inhibition percentage of each drug group was calculated by comparing it with the blank control group. Pain inhibition percentage (%) = (number of writhing times in the blank control group - number of writhing times in the drug group) / number of writhing times in the blank control group × 100%.

[0126] The experimental results are shown in Table 3:

[0127] Table 3 Effects of new diterpenoid alkaloids from Delphinium tricholoma on acetic acid-induced pain in mice

[0128]

[0129] Compared with the blank control group, ***P<0.001.

[0130] To further investigate the analgesic effects of delphinidin A, B, D, G, and delphinidin A / B, new diterpenoid alkaloids isolated from the flowers of Delphinium tricholoma, this study established a pain model in mice by intraperitoneal injection of acetic acid. This simulated pain state allowed for comprehensive evaluation of their analgesic efficacy. The results showed that all six new diterpenoid alkaloids isolated from Delphinium tricholoma significantly reduced the number of writhings in pain-induced mice, demonstrating strong analgesic activity, with writhing inhibition rates exceeding 60%.

[0131] In summary, the results of this study provide a solid experimental basis for further exploring the possibility of these compounds as potential clinical analgesics.

Claims

1. A diterpene alkaloid compound isolated from the flower of Delphinium tricholoma, characterized in that The structural formula of the compound is: in: Compound 1 is named Delphinidinium tricholoma A; Compound 2 is named Delphinium tricholoma B; Compound 3 is named Delphinium tricholoma D; Compound 4 is named Delphinidinium tricholoma G; Compound 5 is named delphinidin A; Compound 6 is named delphinidin B; Compounds 5 and 6 are a pair of regioisomeric mixtures.

2. The method for isolating diterpenoid alkaloid compounds from Delphinium tricholoma flowers according to claim 1, characterized in that Follow these steps: a. Grind the whole plant of Delphinium tricholoma, extract with 10-95% ethanol aqueous solution, methanol or dichloromethane at room temperature by cold soaking, percolation, heating under reflux or ultrasonic extraction, and concentrate under reduced pressure to recover the solvent to obtain an extract; b. Dispersing the extract obtained in step a with 1-5% by mass sulfuric acid or hydrochloric acid, adjusting the pH of the obtained acidic aqueous layer to 9-12 with anhydrous sodium carbonate, aqueous ammonia or sodium hydroxide, and then extracting with organic solvents such as chloroform, ethyl acetate or n-butanol, and concentrating under reduced pressure to recover the organic solvent to obtain total alkaloids; c. Separating the total alkaloids obtained in step b by two, three or four methods selected from the group consisting of normal phase silica gel column chromatography, reverse phase silica gel column chromatography, reverse phase column chromatography, Sephadex LH-20 or semi-preparative high performance liquid chromatography; d. Detect and analyze the product using chromatography or high performance liquid chromatography to obtain new diterpenoid alkaloid compounds.

3. The method for preparing diterpenoid alkaloid compounds from Delphinium tricholoma flowers according to claim 2, characterized in that Two separation methods in step c: Using a combination of normal phase silica gel column chromatography and semi-preparative high performance liquid chromatography, or using a combination of reverse phase silica gel column chromatography and semi-preparative high performance liquid chromatography: The total alkaloid extract obtained was separated by silica gel column chromatography using gradient elution with dichloromethane and methanol in a volume ratio of 100:1, 50:1, 20:1, 10:1, and 1:1 or methanol-water in a volume ratio of 10:90-100:

0. The fractions were analyzed by silica gel thin layer chromatography, and the same fractions were combined to obtain 9 components AI; The obtained fraction D was further separated by normal-phase silica gel column chromatography or reverse-phase silica gel column chromatography using a gradient elution with dichloromethane and methanol in a volume ratio of 100:1, 50:1, 20:1, 10:1, and 1:1 or methanol-water in a volume ratio of 10:90-100:

0. After TLC analysis, the same fractions were combined to obtain compound delphinidinium tricholoma B and a secondary fraction Fr.D10. Fr.D10 was separated by normal phase silica gel column chromatography or reverse phase silica gel column chromatography, with isocratic elution using petroleum ether-acetone in a volume ratio of 5:1 or gradient elution using methanol-water in a volume ratio of 40:60-100:

0. After TLC analysis, the same fractions were combined to obtain Fr.D10e. Fr.D10e was purified by semi-preparative high performance liquid chromatography, with isocratic elution using acetonitrile-water in a volume ratio of 28:72 and 0.1% formic acid to obtain compound delphinidinium tricholometasum G. The obtained fraction E was separated by normal-phase silica gel column chromatography or reverse-phase silica gel column chromatography, using a gradient elution method with petroleum ether-acetone in a volume ratio of 10:1, 5:1, or 3:1, or methanol-water in a volume ratio of 30:70-100:

0. The obtained fractions were analyzed by TLC and then combined to obtain the Fr.E42 fraction. Fr.E42 was purified by semi-preparative high-performance liquid chromatography, using acetonitrile-water in a volume ratio of 24:76 and 0.1% formic acid as the isocratic elution method to obtain the compounds delphinidin A / B, which are a mixture of a pair of regioisomers. The obtained F fraction was separated by normal phase silica gel column chromatography or reverse phase silica gel column chromatography, and gradient eluted with chloroform-methanol with a volume ratio of 70:1, 60:1, 50:1, 20:1, 9:1, 8:2, 7:3, 6:4, 5:5 or methanol-water with a volume ratio of 10:90-100:

0. The obtained fractions were detected by TLC and the same parts were combined to obtain Fr.F1-Fr.F11 fractions; the Fr.F4 fraction was separated by normal phase silica gel column chromatography. Separate the product by column chromatography or reverse-phase silica gel column chromatography, using gradient elution with chloroform-methanol in volume ratios of 25:1, 20:1, and 15:1 or methanol-water in volume ratios of 40:60-100:

0. The resulting fractions are analyzed by TLC and then combined to obtain fraction Fr.F4a. Fr.F4a is purified by semi-preparative high-performance liquid chromatography, using isocratic elution with acetonitrile-water in a volume ratio of 35:65 and 0.1% formic acid to obtain a new compound, delphinidinium tricholometasum D. The obtained fraction I was separated and purified by normal phase silica gel column chromatography or reverse phase silica gel column chromatography, and gradient eluted with chloroform-methanol with a volume ratio of 50:1, 20:1, 9:1, 8:2, 7:3, 6:4, 5:5 or methanol-water with a volume ratio of 10:90-100:

0. After TLC detection, the same fractions were combined to obtain the secondary fraction Fr.I1; Fr.I1 was further separated by normal phase silica gel column chromatography or reverse phase silica gel column chromatography. Purification was carried out by gradient elution with chloroform-methanol in a volume ratio of 20:1, 9:1, 8:2, 7:3, 6:4, and 5:5 or methanol-water in a volume ratio of 40:60-100:

0. After TLC detection, the same fractions were combined to obtain Fr.I4a. Fr.I4a was purified by semi-preparative high-performance liquid chromatography and isocratically eluted with acetonitrile-water in a volume ratio of 23:77 and 0.1% formic acid to obtain a new compound, Delphinidinium tricholometaphyllum A.

4. The method for preparing diterpenoid alkaloid compounds from Delphinium tricholoma flowers according to claim 2, characterized in that Three separation methods in step c: Using a combination of normal phase silica gel column chromatography, reverse phase silica gel column chromatography and semi-preparative high performance liquid chromatography, or using a combination of reverse phase silica gel column chromatography, normal phase silica gel column chromatography and semi-preparative high performance liquid chromatography: The obtained total alkaloid extract was separated by normal phase silica gel column chromatography or reverse phase silica gel column chromatography, using a gradient elution with dichloromethane and methanol in a volume ratio of 100:1, 50:1, 20:1, 10:1, and 1:1 or methanol-water in a volume ratio of 10:90-100:0 as the eluent. The fractions were analyzed by TLC, and the same fractions were combined to obtain 9-11 components AI; The obtained fraction D was separated and purified by reverse phase silica gel column chromatography or normal phase silica gel column chromatography, using a gradient elution with methanol-water in a volume ratio of 10:90-100:0 or sequentially with dichloromethane and methanol in a volume ratio of 100:1, 50:1, 20:1, 10:1, and 1:

1. After TLC detection, the same fractions were combined to obtain a new compound delphinidin B and a secondary fraction Fr.D10. Fr.D10 was separated by normal phase silica gel column chromatography or reverse phase silica gel column chromatography, using isocratic elution with petroleum ether-acetone in a volume ratio of 5:1 or methanol-water in a volume ratio of 10:90-100:

0. After TLC detection, the same fractions were combined to obtain a fraction Fr.D10e. Fr.D10e was purified by semi-preparative high performance liquid chromatography using isocratic elution with acetonitrile-water in a volume ratio of 28:72 and 0.1% formic acid to obtain a new compound delphinidin G. The obtained E was separated by reverse phase silica gel column chromatography or normal phase silica gel column chromatography, sequentially eluting with a gradient of methanol-water in a volume ratio of 30:70-100:0 or petroleum ether-acetone in a volume ratio of 10:1, 5:1, and 3:

1. The obtained fractions were analyzed by TLC and then combined to obtain fraction Fr.E42. The Fr.E42 fraction was purified by semi-preparative high performance liquid chromatography, isocratically eluting with acetonitrile-water in a volume ratio of 24:76 and 0.1% formic acid to obtain compounds delphinidin A / B, which are a mixture of a pair of regioisomers. The obtained F fraction was separated by reverse phase silica gel column chromatography or normal phase silica gel column chromatography, and gradient eluted with methanol-water in a volume ratio of 10:90-100:0 or chloroform-methanol in a volume ratio of 70:1, 60:1, 50:1, 20:1, 9:1, 8:2, 7:3, 6:4, 5:

5. The obtained fractions were detected by TLC and the same parts were combined to obtain fraction Fr.F4. The Fr.F4 fraction was separated by normal phase silica gel column chromatography. The product is separated by chromatography or reverse-phase silica gel column chromatography using a gradient elution with chloroform-methanol in a volume ratio of 25:1, 20:1, or 15:1 or methanol-water in a volume ratio of 40:60-100:

0. The resulting fractions are analyzed by TLC and then combined to obtain fraction Fr.F4a. Fr.F4a is purified by semi-preparative high-performance liquid chromatography using an isocratic elution with acetonitrile-water in a volume ratio of 35:65 and 0.1% formic acid to obtain a new compound, Delphinidinium tricholometaphyllum D. The obtained fraction I was separated by reverse phase silica gel column chromatography or normal phase silica gel column chromatography, and gradient eluted with methanol-water in a volume ratio of 10:90-100:0 or chloroform-methanol in a volume ratio of 50:1, 20:1, 9:1, 8:2, 7:3, 6:4, 5:

5. After TLC detection, the same fractions were combined to obtain a secondary fraction Fr.I4; Fr.I4 was separated by normal phase silica gel column chromatography or reverse phase silica gel column chromatography. Purification was performed by gradient elution using chloroform-methanol with a volume ratio of 20:1, 9:1, 8:2, 7:3, 6:4, and 5:5 or methanol-water with a volume ratio of 40:60-100:

0. After TLC detection, the same fractions were combined to obtain Fr.I4a. Fr.I4a was purified by semi-preparative high-performance liquid chromatography using acetonitrile-water with a volume ratio of 23:77 and 0.1% formic acid as the isocratic eluent to obtain a new compound, Delphinidinium tricholometaphyllum A.

5. The method for preparing diterpenoid alkaloid compounds from Delphinium tricholoma flowers according to claim 2, characterized in that Four separation methods in step c: Using a combination of normal phase silica gel column chromatography, reverse phase silica gel column chromatography, Sephadex LH-20 and semi-preparative high performance liquid chromatography, or using a combination of reverse phase silica gel column chromatography, reverse phase column, Sephadex LH-20 and semi-preparative high performance liquid chromatography: The total alkaloid extract obtained was separated by normal phase silica gel column chromatography or reverse phase silica gel column chromatography, and gradient eluted with dichloromethane and methanol in a volume ratio of 100:1, 50:1, 20:1, 10:1, and 1:1, or with methanol-water in a volume ratio of 10:90-100:

0. The fractions were analyzed by normal phase silica gel thin layer chromatography, and the same fractions were combined to obtain 9 components AI; The obtained fraction D was separated and purified using a reverse phase column or Sephadex LH-20, and gradient or isocratic elution was performed with methanol-water in a volume ratio of 10:90-100:0 or dichloromethane-methanol in a volume ratio of 1:

1. After TLC detection, the same fractions were combined to obtain a new compound delphinidin B and a secondary fraction Fr.D10. Fr.D10 was separated and purified using Sephadex LH-20 column chromatography or a reverse phase column, and isocratic or gradient elution was performed with dichloromethane-methanol in a volume ratio of 1:1 or methanol-water in a volume ratio of 40:60-100:

0. After TLC detection, the same fractions were combined to obtain a fraction Fr.D10e. Fr.D10e was purified by semi-preparative high performance liquid chromatography, and isocratic elution was performed with acetonitrile-water in a volume ratio of 28:72, 0.1% formic acid to obtain a new compound delphinidin G. The obtained fraction E is separated using Sephadex LH-20 or a reverse phase column and isocratically or gradiently eluted with dichloromethane-methanol in a volume ratio of 1:1 or methanol-water in a volume ratio of 30:70-100:

0. The obtained fractions are analyzed by TLC and then combined to obtain fraction Fr.E42. Fr.E42 is purified by semi-preparative high performance liquid chromatography and isocratically eluted with acetonitrile-water in a volume ratio of 24:76 and 0.1% formic acid to obtain compounds delphinidin A / B, which are a mixture of a pair of regioisomers. The obtained F fraction was separated by normal phase silica gel column chromatography or reverse phase silica gel column chromatography, and gradient eluted with chloroform-methanol with a volume ratio of 70:1, 60:1, 50:1, 20:1, 9:1, 8:2, 7:3, 6:4, 5:5 or methanol-water with a volume ratio of 10:90-100:

0. The obtained fractions were detected by TLC and the same parts were combined to obtain the Fr.F4 fraction; the Fr.F4 fraction was separated by elution. Separate using Sephadex LH-20 or reverse-phase column chromatography, isocratically eluting with a 1:1 volume ratio of dichloromethane-methanol or a 40:60-100:0 volume ratio of methanol-water. After TLC analysis, combine the same fractions to obtain fraction Fr.F4a. Fr.F4a is purified by semi-preparative HPLC, isocratically eluting with a 35:65 volume ratio of acetonitrile-water, 0.1% formic acid, to obtain a new compound, delphinidinium tricholometasum D. The obtained fraction I was separated and purified using a reverse phase column or Sephadex LH-20, and gradient or isocratic elution was performed with methanol-water or pure methanol in a volume ratio of 40:60-100:

0. After TLC detection, the same fractions were combined to obtain a secondary fraction Fr.I4; Fr.I4 was separated and purified using Sephadex LH-20 or a reverse phase column, and isocratic or gradient elution was performed with methanol or methanol-water in a volume ratio of 40:60-100:

0. After TLC detection, the same fractions were combined to obtain a fraction Fr.I4a; Fr.I4a was purified by semi-preparative high performance liquid chromatography, and isocratic elution was performed with acetonitrile-water in a volume ratio of 23:77 and 0.1% formic acid to obtain a new compound delphinidinium tricholometaphyllum A.

6. Use of the diterpene alkaloid compound isolated from the flower of Delphinium tricholoma according to claim 1 in the preparation of analgesic drugs.

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