Diterpenoid alkaloid compounds in Delphinium aemulans and preparation method and use thereof

Abstract

The present invention relates to diterpenoid alkaloid compounds in Delphinium aemulans Nevski var. aemulans and a preparation method and use thereof. The compounds use Delphinium aemulans Nevski var. aemulans as a raw material, are extracted with a solvent, treated with acid and base, subjected to solvent extraction, and separated by two, three or four of silica gel column chromatography, Sephadex LH-20 column chromatography, preparative thin layer chromatography or high performance liquid chromatography, and detected and analyzed by thin layer chromatography or high performance liquid chromatography to obtain two new compounds: Compound 1 named aemulanosine A; Compound 2 named aemulanosine B. The voltage-gated potassium channel activities of the two compounds were measured. The experimental results show that aemulanosine A and aemulanosine B isolated from Delphinium aemulans Nevski var. aemulans show inhibitory effects on voltage-gated potassium channel currents and can be used for the preparation of drugs with the activity of inhibiting voltage-gated potassium channel currents.

Description

Technical Field

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

[0002] Diterpene alkaloids are a class of natural compounds with complex structures and diverse activities. They are mainly found in plants of the genera Aconitum and Delphinium in the Ranunculaceae family. They have multiple biological activities such as anti-arrhythmic and analgesic. Tacheng Delphinium (Delphiniumaemulans Navski.) is a plant of the genus Delphinium in the Ranunculaceae family. It is distributed in northwest Xinjiang, Russia, Kazakhstan and other Central Asian regions, and grows on hillsides at an altitude of 1,400 meters. The plant is quite abundant in Xinjiang and is used by the people for pain relief, wind and cold, and treatment of injuries and sores from falls.

[0003] Ion channels play a vital role in the nervous system, cardiovascular system, and muscular system. They are a type of biological macromolecule necessary for various life activities of the human body and are also important drug targets. At present, the extraction, separation, structural identification, and related pharmacological research of diterpenoid alkaloids from Tacheng Delphinium flowers are still in the initial stage at home and abroad. Therefore, it is of great significance to conduct a systematic and in-depth study on the diterpenoid alkaloid components in Tacheng Delphinium flowers, clarify the material basis of their ion channel activity, and discover new diterpenoid alkaloid compounds with specific activity and high-efficiency and low-toxic drug lead compounds. Summary of the invention

[0004] The purpose of the present invention is to provide a diterpene alkaloid compound from Tacheng Delphinium flower, a preparation method and a use thereof. The compound is extracted from Tacheng Delphinium flower medicinal material with a solvent, and then acid-dissolved and alkali-precipitated, and then solvent-extracted. The compound is separated by two, three or four methods, namely, silica gel column chromatography, preparative thin layer chromatography or dextran gel LH-20 column chromatography, to obtain two new diterpene alkaloid compounds, one of which is named Tacheng Delphinium A; and the other is named Tacheng Delphinium B. The obtained compounds were subjected to an inhibition assay of voltage-gated potassium ion (Kv) channel current. The experimental results showed that the inhibitory effects of Tacheng Delphinium A and B on voltage-gated potassium ion channel current (IKv) were dose-dependent, and the IC values ​​of the two compounds for inhibiting voltage-gated potassium ion channel current (IKv) were 50 The values ​​of 1.5 and 2.0 are 81.80 μM and 8.90 μM respectively. It can be used to prepare drugs for inhibiting voltage-gated potassium channel current (IKv).

[0005] The diterpene alkaloid compound isolated from Tacheng delphinium flower of the present invention has the structural formula:

[0006]

[0007] Wherein:

[0008] The name of Compound 1 is Tacheng delphinine A;

[0009] The name of Compound 2 is Tacheng delphinine B.

[0010] The preparation method of the diterpenoid alkaloid compounds in the above-mentioned Tacheng delphinium flowers is carried out according to the following steps:

[0011] a. After crushing the whole herb of Tacheng delphinium as the raw material, use an ethanol aqueous solution with a volume fraction of 10 - 95%, methanol or chloroform at room temperature, and extract by cold soaking, percolation, heating under reflux or ultrasonic extraction, and then concentrate under reduced pressure to recover the solvent to obtain an extract;

[0012] b. Disperse and treat the extract obtained in step a with sulfuric acid or hydrochloric acid with a mass fraction of 1 - 5%, adjust the pH of the obtained acid aqueous layer to 9 - 12 with anhydrous sodium carbonate, ammonia water or sodium hydroxide, and then extract with an organic solvent such as chloroform, ethyl acetate, ether or n-butanol, and concentrate under reduced pressure to recover the organic solvent to obtain total alkaloids;

[0013] c. Separate the total alkaloids obtained in step b by two, three or four methods among silica gel column chromatography, thin layer chromatography, Sephadex LH-20 column chromatography or high performance liquid chromatography to obtain Compound Tacheng delphinine A and Compound Tacheng delphinine B;

[0014] Among them, for the two separation methods:

[0015] Separate the obtained total alkaloid extract by normal-phase silica gel column chromatography, and use chloroform and methanol with a volume ratio of 100:1, 80:1, 60:1, 40:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1 as the eluent for gradient elution. Collect one fraction every 500 ml, analyze the fractions by silica gel thin layer chromatography, and combine the same fractions to obtain 5 components A - E;

[0016] Continue to further separate the obtained Component A by normal-phase silica gel column chromatography or reverse-phase silica gel column chromatography, and use petroleum ether - acetone + 0.2% diethylamine with a volume ratio of 20:1, 10:1, 5:1, 2:1, 1:1 and 0:1 or methanol and water with a volume ratio of 5:95 - 100:0 as the eluent for gradient elution. After analysis by silica gel thin layer chromatography and high performance liquid chromatography, combine the same fractions to obtain 4 components A-1 to A-4; Component A-3 is purified by semi-preparative high performance liquid chromatography and eluted with an acetonitrile - 0.1% formic acid aqueous solution with a volume ratio of 20:80 - 47:53 to obtain Compound Tacheng delphinine A;

[0017] The obtained fraction C was further separated by normal-phase silica gel column chromatography or reverse-phase silica gel column chromatography, and gradient elution was carried out with petroleum ether-acetone + 0.2% diethylamine with volume ratios of 20:1, 10:1, 5:1, 2:1, 1:1 and 0:1 or methanol and water with volume ratios of 5:95 - 100:0 as the eluent. After silica gel thin-layer chromatography and high-performance liquid chromatography analysis, the same fractions were combined to obtain 4 fractions C-1 to C-4; fraction C-2 was purified by semi-preparative high-performance liquid chromatography, and gradient elution was carried out with acetonitrile-0.1% formic acid aqueous solution with volume ratios of 22:78 - 35:65 to obtain the compound talassaconitine B;

[0018] Three separations:

[0019] The obtained total alkaloid extract was separated by normal-phase silica gel column chromatography or reverse-phase silica gel column chromatography, and gradient elution was carried out with chloroform-methanol with volume ratios of 40:1, 20:1, 10:1, 5:1 and 1:1 or methanol-water with volume ratios of 5:95 - 80:20 as the eluent. One fraction was collected every 500 ml. After silica gel thin-layer chromatography analysis, the same fractions were combined to obtain 5 fractions A - E;

[0020] The obtained fraction A was further separated by reverse-phase silica gel column chromatography or normal-phase silica gel column chromatography, and gradient elution was carried out with methanol-water with volume ratios of 5:95 - 80:20 or petroleum ether-acetone + 0.2% diethylamine with volume ratios of 10:1, 5:1, 2:1, 1:1 and 0:1 as the eluent. After silica gel thin-layer chromatography and high-performance liquid chromatography analysis, the same fractions were combined to obtain 4 fractions A-1 to A-4; fraction A-3 was purified by semi-preparative high-performance liquid chromatography, and then gradient elution was carried out with acetonitrile-0.1% formic acid aqueous solution with volume ratios of 20:80 - 47:53 to obtain the compound talassaconitine A;

[0021] The obtained fraction C was further separated by Sephadex LH-20 gel column chromatography, and isocratic elution was carried out with chloroform-methanol with volume ratios of 1:1 or 0:1. After silica gel thin-layer chromatography and high-performance liquid chromatography analysis, the same fractions were combined to obtain 4 fractions C-1 to C-4; C-2 was purified by semi-preparative high-performance liquid chromatography, and isocratic elution was carried out with acetonitrile-0.1% formic acid with volume ratio of 28:72 to obtain the compound talassaconitine B;

[0022] Four separations:

[0023] The obtained total alkaloid extract was separated by Sephadex LH-20 column chromatography, and isocratic elution was carried out with chloroform-methanol with volume ratios of 1:1 or 0:1 as the solvent system. The fractions were analyzed by silica gel thin-layer chromatography, and the same fractions were combined to obtain 5 fractions A - E;

[0024] Component A was subjected to reversed-phase silica gel column chromatography and eluted with a gradient of methanol-water solution with a volume ratio of 5:95 - 80:20. After analysis by TLC and HPLC, the same fractions were combined to obtain 4 components A-1 to A-4. Component A-3 was purified by semi-preparative high-performance liquid chromatography and eluted with a gradient of acetonitrile - 0.1% formic acid aqueous solution with a volume ratio of 20:80 - 47:53 to obtain the compound talassicumine A;

[0025] The obtained Component C was subjected to normal-phase silica gel column chromatography and eluted with a gradient using petroleum ether - acetone + 0.2% diethylamine with volume ratios of 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1 as the eluent. After analysis by silica gel thin-layer chromatography and high-performance liquid chromatography, the same fractions were combined to obtain 4 components C-1 to C-4; Component C-2 was purified by preparative thin-layer chromatography and developed with a developing agent of chloroform - methanol - water with a volume ratio of 82:16:2 to obtain the compound talassicumine B.

[0026] Use of the diterpenoid alkaloid compounds isolated from Delphinium talassicum in the preparation of drugs for inhibiting voltage-gated potassium channel current activity.

[0027] For the diterpenoid alkaloid compounds, preparation method and use in Delphinium talassicum of the present invention, for the obtained compound talassicumine A and compound talassicumine B, thin-layer chromatography or high-performance liquid chromatography analysis was performed for detection and analysis. The developing systems were mixtures of chloroform and methanol with volume ratios of 20:1, 10:1, 8:1, 5:1, 3:1, 1:1, mixtures of chloroform, methanol and water with volume ratios of 82:16:2 or 84:14:2, and mixtures of petroleum ether saturated with ammonia water and acetone with volume ratios of 10:1, 5:1 or 3:1, and 2 new monomeric diterpenoid alkaloid compounds were obtained.

[0028] For the diterpenoid alkaloid compounds, preparation method and use in Delphinium talassicum of the present invention, the inhibitory determination of Kv channel current was performed on the obtained 2 new diterpenoid alkaloid compounds. The experimental results showed that the diterpenoid alkaloid compounds isolated from Delphinium talassicum have the activity of inhibiting voltage-gated potassium channel current (IKv) and can be used in the preparation of drugs for inhibiting voltage-gated potassium channel current (IKv).

[0029] For the diterpenoid alkaloid compounds, preparation method and use in Delphinium talassicum of the present invention, the compounds use the medicinal materials of Delphinium talassicum as the raw material, are extracted with a solvent, acid-dissolved and alkali-precipitated, and then solvent-extracted, and separated by two, three or four methods of silica gel column chromatography, preparative thin-layer chromatography or Sephadex LH-20 column chromatography to obtain 2 new diterpenoid alkaloid compounds. Compound 1 is named talassicumine A; Compound 2 is named talassicumine B, where:

[0030] Compound 1 is named aemulanine A and is a white needle crystal. Its molecular formula was determined to be C + 25 H 37 NO 8 by HRESI(+)MS (m / z 480.2567 [M+H] 1 , theoretical value 480.2592); its structure was determined based on 13 H, 19 C, two-dimensional nuclear magnetic resonance data and X-ray single crystal diffraction. Compound 1 is a newly discovered C 1 -diterpenoid alkaloid of a new skeleton type, and it was named aemulanine A. Its 13 H and 1 C NMR data assignments are shown in Table 1 [600 MHz ( 13 H), 150 MHz ( 3 C), solvent: CD

[0031] OD]; + Compound 2 is aemulanine B and is a white amorphous powder. Its molecular formula was determined to be C 24 H 35 NO 8 by HRESI(+)MS (m / z 466.2411 [M+H] 1 , theoretical value 466.2435); its structure was determined based on 13 H, 1 C and two-dimensional nuclear magnetic resonance data, and it was named aemulanine B. Its 13 H and 1 C NMR data assignments are shown in Table 1 [600 MHz ( 13 H), 150 MHz ( 3 C), solvent: CD

[0032] Table 1 1 H (600 MHz) and 13 C (150 MHz) NMR data of Compounds 1 and 2 [δ (ppm), J (Hz)]

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the 1 H NMR spectrum of Compound 1 aemulanine A of the present invention;

[0035] Figure 2 is the 13 C NMR spectrum of Compound 1 aemulanine A of the present invention;​

[0036] Figure 3 For compound 2 of the present invention, talassicumine B 1 1H NMR spectrum;

[0037] Figure 4 For compound 2 of the present invention, talassicumine B 13 13C NMR spectrum;

[0038] Figure 5 For the effect of talassicumine A and talassicumine B of the present invention on IKv in H9C2 cells. Detailed implementation manners

[0039] All reagents used are of analytical purity. Acetonitrile in high performance liquid chromatography is of HPLC grade (Merk, USA). Column chromatography silica gel (100 - 200 mesh, 200 - 300 mesh): produced by Qingdao Ocean Chemical Factory; thin layer chromatography silica gel is GF 254 , produced by Yantai Huangwu Silica Gel Development and Test Factory; Sephadex LH-20 gel: produced by Pharmacia, Sweden. High performance liquid chromatography (Agilent, USA): P680 HPLC pump, ASI-100 automatic sampler, TCC-100 column oven, UVD170U ultraviolet detector (four wavelengths), quaternary eluent, online degasser, Chromeleon chromatography workstation. Preparative high performance liquid chromatography (Jiangsu Hanbang Technology Co., Ltd.): NP7005C pump, N3000D ultraviolet detector (two wavelengths), semi-preparative dynamic mixer, EasyChrom-1000 chromatography workstation. Mass spectrometry was determined using a QSTAR Elite mass spectrometer (Applied Biosystems / MDS Sciex); nuclear magnetic resonance was determined using a Varian Vnmrs 600 / 400 type nuclear magnetic resonance spectrometer (Varian, USA); electronic balance (METTER AC-100, Sartorious, Germany); rotary evaporator (N-1001D, Shanghai Ailang Instrument Co., Ltd.); high-precision polarimeter (UAutopol VI, Rudolph, USA);

[0040] Delphinium aemulans Navski was collected in Emin County, Tacheng City, Xinjiang Uygur Autonomous Region, China in July 2015, and was identified as Delphinium aemulans Navski of the genus Delphinium in the family Ranunculaceae by Researcher Feng Ying of the Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences. The specimen is preserved in the Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences.

[0041] Example 1 (Two separations)

[0042] Preparation of diterpenoid alkaloid compounds:

[0043] a. After pulverizing 10.0 kg of the whole herb of Delphinium iliense Huth, soak the medicinal materials thoroughly with a 5% Na 2 CO 3 solution at room temperature for 3 hours, extract by cold soaking with 50 L of chloroform solution, and evaporate the solvent under reduced pressure to obtain the crude extract paste of Delphinium iliense Huth;

[0044] b. Disperse the crude extract paste obtained in step a with a 2% sulfuric acid solution, separate the acidic aqueous layer, adjust the pH to 9 with Na 2 CO 3 , then extract with ethyl acetate, evaporate the ethyl acetate under reduced pressure to obtain total alkaloid extract 1; alkalize the alkaline aqueous layer with 10% NaOH to pH 12, then extract with n-butanol, evaporate the n-butanol under reduced pressure to obtain total alkaloid extract 2, and combine total alkaloid extract 1 and total alkaloid extract 2 to obtain the total alkaloid extract;

[0045] c. Separate the obtained total alkaloid extract by normal-phase silica gel column chromatography, and perform gradient elution with chloroform and methanol with a volume ratio of 100:1, 60:1, 40:1, 20:1, 10:1, 5:1, 3:1, 1:1 as the eluent. Collect one fraction every 500 ml, analyze the fractions by silica gel thin-layer chromatography (TLC), and combine the same fractions to obtain 5 components A - E;

[0046] Continue to further separate the obtained component A by reverse-phase silica gel (ODS) column chromatography, perform gradient elution with a methanol-aqueous solution with a volume ratio of 5:95 - 80:20, combine the same fractions after analysis by silica gel thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) to obtain 4 components A-1 to A-4; Component A-3 is purified by semi-preparative high-performance liquid chromatography, and gradient elution is performed with acetonitrile - 0.1% formic acid water with a volume ratio of 22:78 - 35:65 to obtain the compound aemulanine A;

[0047] Continue to further separate the obtained component C by normal-phase silica gel column chromatography, and perform gradient elution with petroleum ether - acetone + 0.2% diethylamine with a volume ratio of 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1 as the eluent. Combine the same fractions after analysis by silica gel thin-layer chromatography (TLC) and HPLC to obtain 4 components C-1 to C-4; Component C-2 is purified by semi-preparative high-performance liquid chromatography, and gradient elution is performed with acetonitrile - 0.1% formic acid water with a volume ratio of 22:78 - 35:65 to obtain the compound aemulanine B;

[0048] The compounds delavaconitine A and delavaconitine B obtained in step c were analyzed by thin-layer chromatography, developed with chloroform-methanol-water with a volume ratio of 82:16:2 as the developing agent, to determine that 2 new monomeric diterpenoid alkaloid compounds were obtained.

[0049] Example 2 (Two Separations)

[0050] a. After crushing 10.0 kg of the whole herb of Delphinium semenovianum, it was extracted by percolation with 50 L of 80% ethanol by volume at room temperature, and the solvent was evaporated under reduced pressure to obtain the crude extract paste of Delphinium semenovianum.

[0051] b. The crude extract paste obtained in step a was dispersed with 1% sulfuric acid by mass. The obtained acid aqueous layer was adjusted to pH = 10 with ammonia water, and then extracted with chloroform. The chloroform was evaporated under reduced pressure to obtain the total alkaloid extract paste 1; the alkaline aqueous layer was further alkalized to pH 12 with 10% NaOH, and then extracted with n-butanol. The n-butanol was evaporated under reduced pressure to obtain the total alkaloid extract paste 2. The total alkaloid extract paste 1 and the total alkaloid extract paste 2 were combined to obtain the total alkaloid extract paste.

[0052] c. The obtained total alkaloid extract paste was separated by Sephadex LH-20 gel column chromatography, and isocratic elution was carried out with chloroform and methanol with a volume ratio of 1:1 as the eluent. One fraction was collected every 20 ml. The fractions were analyzed by silica gel thin-layer chromatography (TLC), and the same fractions were combined to obtain 5 components A-E.

[0053] The obtained fraction A was further separated by Sephadex LH-20 gel column chromatography with isocratic methanol elution. After analysis by silica gel thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC), the same fractions were combined to obtain 4 fractions A-1 to A-4; fraction A-3 was further separated and purified by preparative thin-layer chromatography, developed with chloroform-methanol with a volume ratio of 20:1 as the developing agent, to obtain the compound delavaconitine A.

[0054] The obtained fraction C was further separated by Sephadex LH-20 gel column chromatography with isocratic methanol elution. After analysis by TLC and HPLC, the same fractions were combined to obtain 4 fractions C-1 to C-4; fraction C-2 was further separated and purified by preparative thin-layer chromatography, developed with petroleum ether-acetone saturated with ammonia water with a volume ratio of 5:1 as the developing agent, to obtain the compound delavaconitine B.

[0055] The compounds delavaconitine A and delavaconitine B obtained in step c were detected and analyzed by high performance liquid chromatography. Gradient elution was carried out with acetonitrile-formic acid aqueous solution with a volume ratio of 10:90 - 80:20 to determine that 2 new monomeric diterpenoid alkaloid compounds were obtained.

[0056] Example 3 (Three Separations)

[0057] a. After pulverizing 10.0 kg of the whole herb of Delphinium iliense Huth, ultrasonic extraction was carried out with methanol, and the methanol was recovered by reduced pressure concentration to obtain the total extract;

[0058] b. The total extract obtained in step a was dispersed in an aqueous hydrochloric acid solution with a mass fraction of 3%, filtered, and the obtained acidic aqueous solution was adjusted to pH = 10 with Na 2 CO 3 and then extracted with ethyl acetate. The ethyl acetate was evaporated under reduced pressure to obtain the total alkaloid extract 1; the alkaline aqueous layer was further alkalized to pH 12 with 10% NaOH, and then extracted with n-butanol. The n-butanol was evaporated under reduced pressure to obtain the total alkaloid extract 2. The total alkaloid extracts 1 and 2 were combined to obtain the total alkaloid extract;

[0059] c. The total alkaloid extract obtained in step b was separated by normal-phase silica gel column chromatography, and gradient elution was carried out successively with chloroform-methanol 60:1, 40:1, 20:1, 10:1, 5:1, and 1:1 as the solvent systems. Each 500 ml was collected as a fraction, and the fractions were analyzed by silica gel thin-layer chromatography (TLC), and the same fractions were combined to obtain 5 fractions (A - E);

[0060] Fraction A was further separated by reverse-phase silica (ODS) column chromatography, and gradient elution was carried out with a methanol-aqueous solution with a volume ratio of 5:95 - 80:20. After analysis by silica gel thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), the same fractions were combined to obtain 4 fractions A-1 to A-4; A-3 was purified by semi-preparative high-performance liquid chromatography, and gradient elution was carried out with an acetonitrile-0.1% formic acid with a volume ratio of 10:90 - 40:60 to obtain the compound delavaconitine A;

[0061] Fraction C was separated by Sephadex LH-20 gel column chromatography, and isocratic elution was carried out with methanol. After analysis by silica gel thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), the same fractions were combined to obtain 4 fractions C-1 to C-4; C-2 was purified by semi-preparative high-performance liquid chromatography, and gradient elution was carried out with an acetonitrile-0.1% formic acid with a volume ratio of 22:78 - 35:65 to obtain the compound delavaconitine B;

[0062] The compound delavaconitine A and delavaconitine B obtained in step c were analyzed by thin-layer chromatography, and developed with a petroleum ether-acetone saturated with ammonia water with a volume ratio of 5:1 as the developing agent to determine 2 new diterpenoid alkaloid monomer compounds.

[0063] Example 4 (Three Separations)

[0064] a. After pulverizing 10.0 kg of the whole herb of Delphinium iliense Huth, ultrasonic extraction was carried out with 50 L of chloroform at room temperature, and the solvent was evaporated under reduced pressure to obtain the crude extract of Delphinium iliense Huth;

[0065] b. Disperse the crude extract paste obtained in step a with hydrochloric acid with a mass fraction of 4%, adjust the pH of the resulting acidic aqueous solution to 10 with ammonia water, then extract with ethyl acetate, and evaporate the ethyl acetate under reduced pressure to obtain the total alkaloid paste 1; alkalize the alkaline water layer with 10% NaOH to a pH value of 12, then extract with n-butanol, and evaporate the n-butanol under reduced pressure to obtain the total alkaloid paste 2. Combine the total alkaloid paste 1 and the total alkaloid paste 2 to obtain the total alkaloid paste;

[0066] c. Subject the total alkaloid paste obtained in step b to reverse-phase silica gel (ODS) column chromatography separation, elute with a methanol-aqueous solution gradient with a volume ratio of 5:95 - 80:20, collect one fraction every 500 ml, and combine the same fractions after silica gel thin-layer chromatography (TLC) analysis to obtain 5 fractions A - E;

[0067] Fraction A is separated by Sephadex LH-20 gel column chromatography and eluted isocratically with methanol. Combine the same fractions after silica gel thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analysis to obtain 4 fractions A-1 to A-4; A-3 is purified by preparative thin-layer chromatography and developed with a petroleum ether-acetone solution saturated with ammonia water with a volume ratio of 5:1 to obtain the compound talassicumine A;

[0068] Fraction C is separated by Sephadex LH-20 gel column chromatography and eluted isocratically with methanol. Combine the same fractions after silica gel thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analysis to obtain 4 fractions C-1 to C-4; C-2 is purified by preparative thin-layer chromatography and developed with a chloroform-methanol-water solution with a volume ratio of 82:16:2 to obtain the compound talassicumine B;

[0069] Detect and analyze the compound talassicumine A and talassicumine B obtained in step c by high-performance liquid chromatography analysis, and perform gradient elution with an acetonitrile-formic acid aqueous solution with a volume ratio of 25:75 - 80:20 to determine 2 new diterpenoid alkaloid monomer compounds.

[0070] Example 5 (Four Separations)

[0071] a. Take 10.0 kg of the whole herb of Delphinium talassicum, crush it, and reflux extract it with 50 L of 80% ethanol at room temperature. Evaporate the solvent under reduced pressure to obtain the crude extract paste of Delphinium talassicum;

[0072] b. Disperse the crude extract paste obtained in step a with sulfuric acid with a mass fraction of 2%, adjust the pH value of the resulting acidic aqueous solution to 12 with 5% NaOH, extract with n-butanol, and evaporate the n-butanol under reduced pressure to obtain the total alkaloid paste;

[0073] c. The obtained total alkaloid extract was separated by Sephadex LH-20 gel column chromatography, and isocratic elution was carried out with a chloroform-methanol mixture with a volume ratio of 1:1 as the eluent. One fraction was collected every 20 ml. The fractions were analyzed by silica gel thin layer chromatography (TLC), and the same fractions were combined to obtain 5 components A-E;

[0074] Component A was further separated by reversed-phase silica gel (ODS) column method, and gradient elution was carried out with a methanol-aqueous solution with a volume ratio of 5:95 - 80:20. After analysis by silica gel thin layer chromatography (TLC) and high performance liquid chromatography (HPLC), the same fractions were combined to obtain 4 components A-1 to A-4; A-3 was purified by preparative thin layer chromatography, developed with a chloroform-methanol mixture with a volume ratio of 10:1 as the developing agent, and compound Tacheng delphinine A was obtained;

[0075] Component C was further separated by normal-phase silica gel column chromatography, and gradient elution was carried out with a petroleum ether-acetone + 0.2% diethylamine mixture with volume ratios of 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1 as the eluent. After analysis by silica gel thin layer chromatography (TLC) and high performance liquid chromatography (HPLC), the same fractions were combined to obtain 4 components C-1 to C-4; Component C-2 was purified by semi-preparative high performance liquid chromatography, with gradient elution of acetonitrile-0.1% formic acid with a volume ratio of 22:78 - 35:65, and compound Tacheng delphinine B was obtained;

[0076] Compound Tacheng delphinine A and Tacheng delphinine B obtained in step c were analyzed by thin layer chromatography, developed with a chloroform-methanol-water mixture with a volume ratio of 82:16:2 as the developing agent, to determine that 2 new diterpenoid alkaloid monomer compounds were obtained.

[0077] Example 6 (Four Separations)

[0078] a. 10.0 kg of the whole herb of Delphinium semenovianum Regel was crushed and ultrasonically extracted with 50 L of methanol at room temperature. The solvent was evaporated under reduced pressure to obtain the crude extract of Delphinium semenovianum Regel;

[0079] b. The crude extract obtained in step a was dispersed with 2% sulfuric acid by mass fraction. The obtained acidic aqueous solution was adjusted to pH 11 with 5% NaOH, extracted with n-butanol, and the n-butanol was evaporated under reduced pressure to obtain the total alkaloid extract;

[0080] c. The total alkaloid extract obtained in step b was separated by normal-phase silica gel column chromatography, and gradient elution was carried out successively with chloroform-methanol mixtures with volume ratios of 60:1, 40:1, 20:1, 10:1, 5:1, and 1:1 as the solvent system. One fraction was collected every 500 ml. The fractions were analyzed by silica gel thin layer chromatography (TLC), and the same fractions were combined to obtain 5 segments of components A-E;

[0081] Component A was further separated by reverse-phase silica gel (ODS) column chromatography and eluted with a gradient of methanol-water solution with a volume ratio of 5:95 - 80:20. After analysis by silica gel thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), the same fractions were combined to obtain 4 components A-1 to A-4; Component A-3 was purified by semi-preparative high-performance liquid chromatography and eluted with a gradient of acetonitrile-0.1% formic acid water with a volume ratio of 20:80 - 47:53 to obtain the compound aemulanine A;

[0082] Component C was separated by Sephadex LH-20 gel column chromatography and eluted isocratically with methanol. After analysis by silica gel thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), the same fractions were combined to obtain 4 components C-1 to C-4; Component C-2 was purified by preparative thin-layer chromatography and developed with a developing agent of chloroform-methanol-water with a volume ratio of 82:16:2 to obtain the compound aemulanine B.

[0083] Example 7

[0084] The use of the diterpenoid alkaloid compounds isolated from Delphinium aemulans in ion channel activity, taking the mouse cardiomyocyte cell line H9C2 as an example:

[0085] Screening for the ion channel activity of the obtained diterpenoid alkaloid compounds:

[0086] Experimental cells:

[0087] Mouse cardiomyocyte cell line H9C2: Cell Bank of Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences;

[0088] Experimental instruments and reagents:

[0089] Four-channel automatic patch clamp Patchliner (Germany, Nion), dimethyl sulfoxide (Merck);

[0090] Experimental content:

[0091] Test drug working solution: Prepare stock solutions of aemulanine A and aemulanine B at 200 mM with dimethyl sulfoxide (DMSO), and then serially dilute them with extracellular fluid to 6 concentration gradients of 5, 10, 30, 50, 100, and 300 μM. The blank control group was the solvent control, and 1.5 μL of dimethyl sulfoxide was prepared in 998.5 μL of extracellular fluid and mixed well; the above-prepared solutions were used immediately and stored at room temperature in the dark;

[0092] Cell culture: Mouse cardiomyocyte cell line H9C2. The complete medium was prepared in a ratio of 90% DMEM + 10% FBS + 100 mg / mL streptomycin and 100 U / mL penicillin. Gently mix and store in a 4°C refrigerator. When the cells are in good growth condition and the density reaches about 80%, digest with 0.25% trypsin, centrifuge at 800 rpm for 3 min, collect the cells, add 0.5 mL of External Standard solution according to the cell amount, gently mix, and then perform on-machine detection;

[0093] Total current-voltage relationship curve stimulation program of Kv ions:

[0094] The inhibitory effects of talassaconitine A and talassaconitine B on the voltage-gated potassium ion channel current (IKv) in mouse cardiomyocyte H9C2 cells were recorded by whole-cell patch clamp electrophysiology; the cell clamping voltage was -80 mV, and depolarizing pulse stimulation was given in steps of 20 mV from -80 mV to +80 mV, with a total of 17 steps, to induce potassium current. The entire stimulation program was repeated every 10 s, and each stimulation voltage was applied for 50 ms; Patchmaster software was used for current recording and acquisition, and the recording temperature was room temperature (23°C - 25°C); The experimental results are shown in Table 2 and Figure 5 as follows:

[0095] Table 2 Inhibitory effects of two new diterpenoid alkaloid compounds in Delphinium semenovianum Regel on Kv channel current

[0096]

[0097] A: The IC + of the inhibitory effect of talassaconitine A on the total K 50 current was obtained by fitting with the Logistic equation and was 81.80 μM.

[0098] B: Representative diagram of the inhibitory effect of talassaconitine A on the total K + current;

[0099] C: The IC + of the inhibitory effect of talassaconitine B on the total K 50 current was obtained by fitting with the Logistic equation and was 8.90 μM.

[0100] D: Representative diagram of the inhibitory effect of talassaconitine B on the total K + current;

[0101] Table 2 and Figure 5The results showed that the inhibition rates of talcaconitine A and talcaconitine B at 50 μM on the voltage-gated potassium channel current (IKv) were 12.21% and 58.08% respectively, and the inhibitory effects of talcaconitine A and talcaconitine B on IKv showed a dose-dependent trend. The IC 50 values of the two compounds on the voltage-gated potassium channel current (IKv) were 81.80 μM and 8.90 μM respectively. It was suggested that talcaconitine A and B had the activity of inhibiting the voltage-gated potassium channel current (IKv).

Claims

1. A diterpene alkaloid compound isolated from Tacheng Delphinium flower, characterized in that The structural formula of the compound is: in: Compound 1 is named Tacheng delphinium A; The name of compound 2 is Tacheng delphinium B.

2. The method for preparing diterpene alkaloid compounds from Tacheng Delphinium flower according to claim 1, characterized in that Follow these steps: a. After the raw material Tacheng Delphinium flower is crushed, it is extracted with 10-95% ethanol aqueous solution, methanol or chloroform by cold soaking, percolation, heating reflux or ultrasonic extraction at room temperature, and the solvent is recovered by vacuum concentration to obtain an extract; b. The extract obtained in step a is dispersed with 1-5% by mass sulfuric acid or hydrochloric acid, and the obtained acid water layer is adjusted to pH 9-12 with anhydrous sodium carbonate, ammonia water or sodium hydroxide, and then extracted with an organic solvent such as chloroform, ethyl acetate, ether or n-butanol, and concentrated under reduced pressure to recover the organic solvent to obtain total alkaloids; c. Separate the total alkaloids obtained in step b by two, three or four of the following methods: silica gel column chromatography, thin layer chromatography, dextran gel LH-20 column chromatography or high performance liquid chromatography to obtain compound Tacheng delphinium A and compound Tacheng delphinium B.

3. The method for preparing diterpene alkaloid compounds from Tacheng Delphinium flower according to claim 2, characterized in that The two separations in step c are carried out according to the following steps: The obtained total alkaloid extract was separated by normal phase silica gel column chromatography, using chloroform and methanol in a volume ratio of 100:1, 80:1, 60:1, 40:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1 as eluents for gradient elution, collecting one fraction per 500 ml, analyzing the fractions by silica gel thin layer chromatography, and combining the same fractions to obtain 5 components AE; The obtained component A is further separated by normal phase silica gel column chromatography or reverse phase silica gel column chromatography, and gradient elution is performed using petroleum ether-acetone + 0.2% diethylamine in a volume ratio of 20:1, 10:1, 5:1, 2:1, 1:1 and 0:1 or methanol and water in a volume ratio of 5:95-100:0 as eluents. After silica gel thin layer chromatography and high performance liquid chromatography analysis, the same fractions are combined to obtain four components A-1 to A-4; component A-3 is purified by semi-preparative high performance liquid chromatography, and gradient eluted with acetonitrile-0.1% formic acid aqueous solution in a volume ratio of 20:80-47:53 to obtain compound Tacheng delphinium A; The obtained component C is further separated by normal phase silica gel column chromatography or reverse phase silica gel column chromatography, and gradient elution is performed using petroleum ether-acetone + 0.2% diethylamine in volume ratios of 20:1, 10:1, 5:1, 2:1, 1:1 and 0:1 or methanol and water in volume ratios of 5:95-100:0 as eluents. After silica gel thin layer chromatography and high performance liquid chromatography analysis, the same fractions are combined to obtain 4 components C-1 to C-4; component C-2 is purified by semi-preparative high performance liquid chromatography, and gradient elution is performed using acetonitrile-0.1% formic acid aqueous solution in volume ratios of 22:78-35:65 to obtain compound Tacheng delphinium B.

4. The method for preparing diterpene alkaloid compounds from Tacheng Delphinium flower according to claim 2, characterized in that The three separations in step c are carried out according to the following steps: The obtained total alkaloid extract was separated by normal phase silica gel column chromatography or reverse phase silica gel column chromatography, using chloroform-methanol with a volume ratio of 40:1, 20:1, 10:1, 5:1 and 1:1 as eluent or methanol-water with a volume ratio of 5:95-80:20 as eluent for gradient elution, collecting one fraction for every 500 ml, and analyzing by silica gel thin layer chromatography, combining the same fractions to obtain 5 fractions AE; The obtained component A is further separated by reverse phase silica gel column chromatography or normal phase silica gel column chromatography, and gradient elution is performed using methanol-water with a volume ratio of 5:95-80:20 or petroleum ether-acetone + 0.2% diethylamine with a volume ratio of 10:1, 5:1, 2:1, 1:1 and 0:1 as the eluent, and the same fractions are combined after silica gel thin layer chromatography and high performance liquid chromatography analysis to obtain four components A-1 to A-4; component A-3 is purified by semi-preparative high performance liquid chromatography, and then gradient eluted with acetonitrile-0.1% formic acid aqueous solution with a volume ratio of 20:80-47:53 to obtain compound Tacheng delphinium A; The obtained group C was further separated by dextran LH-20 gel column chromatography, isocratically eluted with chloroform-methanol in a volume ratio of 1:1 or 0:1, and the same fractions were combined after silica gel thin layer chromatography and high performance liquid chromatography analysis to obtain 4 components C-1 to C-4; C-2 was purified by semi-preparative high performance liquid chromatography, isocratically eluted with acetonitrile-0.1% formic acid in a volume ratio of 28:72 to obtain compound Tacheng delphinium B.

5. The method for preparing diterpene alkaloid compounds from Tacheng Delphinium flower according to claim 2, characterized in that The four separations in step c are carried out according to the following steps: The total alkaloid extract obtained was separated by Sephadex LH-20 column chromatography, and isocratically eluted using chloroform-methanol with a volume ratio of 1:1 or 0:1 as a solvent system. The fractions were analyzed by silica gel thin layer chromatography, and the same fractions were combined to obtain 5 fractions AE; Component A was subjected to reverse phase silica gel column chromatography, and gradient eluted with methanol-water solution in a volume ratio of 5:95-80:

20. After TLC and HPLC analysis, the same fractions were combined to obtain four components A-1 to A-4. Component A-3 was purified by semi-preparative high performance liquid chromatography, and gradient eluted with acetonitrile-0.1% formic acid aqueous solution in a volume ratio of 20:80-47:53 to obtain compound Tacheng delphinium A. The obtained component C was chromatographed on a normal phase silica gel column using petroleum ether-acetone + 0.2% diethylamine in a volume ratio of 20:1, 10:1, 5:1, 2:1, 1:1 and 0:1 as eluents for gradient elution. After silica gel thin layer chromatography and high performance liquid chromatography analysis, the same fractions were combined to obtain four components C-1 to C-4; component C-2 was purified by preparative thin layer chromatography using chloroform-methanol-water in a volume ratio of 82:16:2 as a developing solvent to obtain compound Tacheng delphinium B.

6. Use of the diterpene alkaloid compound isolated from Tacheng Delphinium flower according to claim 1 in the preparation of a drug for inhibiting the current activity of voltage-gated potassium ion channels.

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