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

Abstract

The invention relates to a diterpenoid alkaloid compound in delphinium ili as well as a preparation method and application thereof.The diterpenoid alkaloid compound is prepared from a delphinium ili medicinal material serving as a raw material through solvent extraction, acid dissolution and alkali precipitation and solvent extraction, the method comprises the following steps: carrying out separation in two, three or four modes of silica gel column chromatography, semi-preparative thin layer chromatography or sephadex LH-20 column chromatography to obtain four new diterpenoid alkaloid compounds 1, which are named as ili delphinidine A; the name of the compound 2 is ili delphinidine B; the name of the compound 3 is ili delphinidine C; and the name of the compound 4 is Ili delphinidine D. The obtained compounds are subjected to analgesic activity determination, and experimental results show that the compounds Iildelphinidine C and D show obvious analgesic activity in an acetic acid-induced mouse writhing model, and can be used for preparing analgesic drugs.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to diterpenoid alkaloid compounds in Delphinium iliense Huth, a preparation method thereof, and uses thereof. Background Art

[0002] Diterpenoid alkaloids are a class of nitrogen-containing natural organic compounds with complex structures and diverse activities. They are mainly present in plants of the genera Aconitum and Delphinium in the Ranunculaceae family. Due to their diverse biological activities and medicinal values, especially their significant effects in anti-arrhythmia and analgesia, they have become research objects that have received extensive attention. Currently, 4 diterpenoid alkaloids have been developed into drugs and used for clinical treatment. So far, more than 1,600 natural diterpenoid alkaloids have been reported, which are mainly divided into four categories: C 18 -, C 19 -, C 20 -, and bis-diterpenoid alkaloids. Due to their complex and variable structures, they have the characteristics of coexistence of activity and toxicity, which makes the search for novel-structured, highly effective and low-toxic diterpenoid alkaloid compounds still a hot and difficult point in current research. Delphinium iliense Huth is a perennial herb of the genus Delphinium in the Ranunculaceae family. It is mainly distributed in western Xinjiang, China, growing in mountainous shrubs or rocky slopes at an altitude of about 2,000 meters, and is also distributed in Central Asia. The resources of this plant are quite rich in the Xinjiang region and are mainly used for clearing heat and detoxifying, treating diarrhea, dysentery, rheumatism and enteritis.

[0003] The analgesic activity of diterpenoid alkaloids has been widely studied. Currently, 3-acetylaconitine, lappaconitine and hypaconitine have been developed into non-addictive analgesics for clinical use. Diterpenoid alkaloids are not only the main active ingredients of plants of the genera Aconitum and Delphinium, but also the main source of their toxicity. Therefore, finding lead compounds with good analgesic effects and low toxicity is our main research goal. At present, the extraction, separation, structure identification and related pharmacological research of diterpenoid alkaloids in Delphinium iliense Huth at home and abroad are still in the initial stage. Therefore, it is of great significance to conduct a systematic and in-depth study on the diterpenoid alkaloid components in Delphinium iliense Huth, clarify the material basis of its analgesic activity, discover new diterpenoid alkaloid compounds with specific activities, and highly effective and low-toxic drug lead compounds. Summary of the Invention

[0004] The object of the present invention is to provide diterpenoid alkaloid compounds in Delphinium iliense Huth, a preparation method thereof and uses. The compounds use the medicinal material of Delphinium iliense Huth as the raw material, are extracted with a solvent, acid dissolved and alkali precipitated, and then solvent extracted, and are separated by two, three or four methods among silica gel column chromatography, semi-preparative thin layer chromatography or Sephadex LH-20 column chromatography to obtain 4 new diterpenoid alkaloid compounds. Compound 1 is named delilicine A; Compound 2 is named delilicine B; Compound 3 is named delilicine C; Compound 4 is named delilicine D. And the analgesic activities of the obtained compounds are determined. The experimental results show that compounds delilicine C and D show obvious analgesic activities in the acetic acid-induced writhing model of mice and can be used for preparing analgesic drugs.

[0005] A diterpenoid alkaloid compound isolated from Delphinium iliense Huth according to the present invention, the structural formula of the compound is:

[0006]

[0007] Wherein:

[0008] Compound 1 is named delilicine A;

[0009] Compound 2 is named delilicine B;

[0010] Compound 3 is named delilicine C;

[0011] Compound 4 is named delilicine D.

[0012] The preparation method of the diterpenoid alkaloid compound in Delphinium iliense Huth is carried out according to the following steps:

[0013] a. After the whole herb of the raw material Delphinium iliense Huth is crushed, it is extracted with an ethanol aqueous solution with a volume fraction of 10-95%, absolute methanol or chloroform at room temperature. Absolute methanol and chloroform are subjected to cold soaking, percolation, heating under reflux or ultrasonic extraction, and the solvent is recovered by reduced pressure concentration to obtain an extract;

[0014] b. The extract obtained in step a is dispersed and treated with sulfuric acid or hydrochloric acid with a mass fraction of 1-5%. The obtained acid aqueous layer is adjusted to pH = 9-12 with anhydrous sodium carbonate, ammonia water or sodium hydroxide, and then extracted with an organic solvent chloroform, ethyl acetate or n-butanol, and the organic solvent is recovered by reduced pressure concentration to obtain total alkaloids;

[0015] Alternatively, the crude extract obtained in step a is dispersed and treated with sulfuric acid or hydrochloric acid with a mass fraction of 1-5%, and the resulting acid aqueous solution is adjusted to pH = 10 with ammonia water or anhydrous Na2CO3, and then extracted with ethyl acetate. The ethyl acetate is evaporated under reduced pressure to obtain the total alkaloid extract 1; then the alkaline water layer is further alkalized to pH 12 with 10% NaOH, and then extracted with n-butanol. The n-butanol is evaporated under reduced pressure to obtain the total alkaloid extract 2. The total alkaloid extracts 1 and 2 are combined to obtain the total alkaloid extract;

[0016] c. The total alkaloids obtained in step b are separated by two, three or four methods of silica gel column chromatography, thin layer chromatography, Sephadex LH-20 column chromatography or high performance liquid chromatography, namely, compound 1 delavine A, compound 2 delavine B, compound 3 delavine C, and compound 4 delavine D are obtained.

[0017] For the preparation method of the diterpenoid alkaloid compounds in the above-mentioned Delphinium iliense, the two separations in step c:

[0018] The total alkaloids obtained in step c are separated by normal-phase silica gel column chromatography, and gradient elution is carried out with dichloromethane 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. One fraction is collected every 500 ml, and the fractions are analyzed by silica gel thin layer chromatography, and the same fractions are combined to obtain 8 components A-H;

[0019] The obtained fraction A is further separated by normal-phase silica gel column chromatography or reverse-phase silica gel column chromatography, and gradient elution is carried out 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 or methanol and water with a volume ratio of 5:95-100:0 as the eluent. After TLC and HPLC analysis, the same fractions are combined to obtain 5 components A-1 to A-5; fraction A-3 is purified by semi-preparative high performance liquid chromatography and isocratically eluted with acetonitrile-0.1% formic acid aqueous solution with a volume ratio of 29:71 to obtain delavine A and delavine B;

[0020] The obtained fraction C is further separated by normal-phase silica gel column chromatography or reverse-phase silica gel column chromatography, and gradient elution is carried out 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 or methanol and water with a volume ratio of 5:95-100:0 as the eluent. After silica gel thin layer chromatography and HPLC analysis, the same fractions are combined to obtain 6 components C-1 to C-6; fraction C-3 is purified by semi-preparative high performance liquid chromatography and gradient eluted with acetonitrile-0.1% formic acid aqueous solution with a volume ratio of 20:80-30:70 to obtain delavine C and delavine D.

[0021] The preparation method of diterpenoid alkaloid compounds in Delphinium iliense Huth, the three separations in step c:

[0022] The total alkaloid extract obtained in step c is separated by normal-phase silica gel column chromatography or reversed-phase silica gel column chromatography, and gradient elution is carried out with chloroform-methanol with a volume ratio of 40:1, 20:1, 10:1, 5:1 and 1:1 or methanol-water with a volume ratio of 5:95 - 80:20 as the eluent. One fraction is collected every 500 ml, and after analysis by silica gel thin-layer chromatography, the same fractions are combined to obtain 6 fractions A - F;

[0023] The obtained fraction A is further separated by reversed-phase silica gel column chromatography or normal-phase silica gel column chromatography, and gradient elution is carried out with methanol-water with a volume ratio of 5:95 - 70:30 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. After analysis by silica gel thin-layer chromatography and HPLC, the same fractions are combined to obtain 5 fractions A-1 to A-5; Fraction 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 - 40:60 to obtain compounds delphatine A and delphatine B;

[0024] The obtained fraction C is further separated by Sephadex LH-20 gel column chromatography and isocratically eluted with chloroform-methanol with a volume ratio of 1:1 or 0:1. After analysis by silica gel thin-layer chromatography and HPLC, the same fractions are combined to obtain 5 fractions C-1 to C-5; C-4 is purified by semi-preparative high performance liquid chromatography and isocratically eluted with acetonitrile-0.1% formic acid with a volume ratio of 28:72 to obtain compounds delphatine C and delphatine D.

[0025] The preparation method of diterpenoid alkaloid compounds in Delphinium iliense Huth, the four separations in step c:

[0026] The total alkaloid extract obtained in step c is separated by Sephadex LH-20 column chromatography and isocratically eluted with chloroform-methanol with a volume ratio of 1:1 or 0:1 as the solvent system. The fractions are analyzed by silica gel thin-layer chromatography, and the same fractions are combined to obtain 9 fractions A - I;

[0027] Fraction A is separated by reversed-phase silica gel column chromatography and gradient eluted with methanol-aqueous solution with a volume ratio of 5:95 - 80:20. After analysis by TLC and HPLC, the same fractions are combined to obtain 4 fractions A-1 to A-4. Fraction A-3 is purified by semi-preparative high performance liquid chromatography and gradient eluted with acetonitrile-0.1% formic acid aqueous solution with a volume ratio of 20:80 - 47:53 to obtain compound delphatine A;

[0028] The obtained fraction C was subjected to normal-phase silica gel column chromatography and gradient elution was performed 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 eluents. After silica gel thin-layer chromatography and HPLC analysis, the same fractions were combined to obtain four fractions C-1 to C-4; fraction C-2 was purified by preparative thin-layer chromatography and developed using chloroform-methanol-water with a volume ratio of 82:16:2 as the developing agent to obtain compound illydeline B;

[0029] The obtained fraction E was subjected to normal-phase silica gel column chromatography and gradient elution was performed using petroleum ether-acetone + 0.2% diethylamine with volume ratios of 10:1, 5:1, 2:1, 1:1, and 0:1 as the eluents. After silica gel thin-layer chromatography and HPLC analysis, the same fractions were combined to obtain five fractions E-1 to E-5; fraction E-3 was purified by semi-preparative high performance liquid chromatography with gradient elution using acetonitrile-0.1% formic acid aqueous solution with volume ratios of 20:80 - 47:53 to obtain compound illydeline C;

[0030] The obtained fraction I was separated by Sephadex LH-20 column chromatography and isocratic elution was performed using chloroform-methanol with a volume ratio 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 four fractions I-1 to I-4; fraction I-4 was purified by semi-preparative high performance liquid chromatography with isocratic elution using acetonitrile-0.1% formic acid aqueous solution with a volume ratio of 28:72 to obtain compound illydeline D.

[0031] Use of the diterpenoid alkaloid compounds isolated from Delphinium iliense Huth in the preparation of analgesic drugs.

[0032] For the diterpenoid alkaloid compounds, preparation method and use of Delphinium iliense Huth described in the present invention, the analgesic activities of the two obtained new diterpenoid alkaloid compounds were determined in mice. The experimental results showed that the new diterpenoid alkaloids 3 and 4 isolated from Delphinium iliense Huth had obvious analgesic effects, and the strength of the analgesic effect showed a certain dose-effect relationship with the dose. In general, the results provided an experimental basis for these compounds as potential clinical analgesics.

[0033] For the diterpenoid alkaloid compounds, preparation method and use of Delphinium iliense Huth described in the present invention, the compounds used the medicinal materials of Delphinium iliense Huth as raw materials, were extracted with solvents, acid-dissolved and alkali-precipitated, 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 four new diterpenoid alkaloid compounds: compound 1 illydeline A; compound 2 illydeline B; compound 3 illydeline C; compound 4 illydeline D, wherein:

[0034] Compound 1 is named Yili delphinidine A, which is a colorless amorphous powder. Its molecular formula was determined to be C + H 25 H 37 NO8 by HRESI(+)MS (m / z 480.2592 [M+H] 1 , theoretical value 480.2592); its structure was determined based on 13 H, 19 C and two-dimensional nuclear magnetic resonance data. Compound 1 is a newly discovered C 1 -diterpenoid alkaloid, which was named Yili delphinidine A (Sinchianidine A). The 13 H and 1 C NMR data assignments are shown in Table 1 [600 MHz ( 13 H), 150 MHz (

[0035] Compound 2 is Yili delphinidine B, a colorless amorphous powder. Its molecular formula was determined to be C + H 24 H 35 NO8 by HRESI(+)MS (m / z 466.2437 [M+H] 1 , theoretical value 466.2435); its structure was determined based on 13 H, 19 C and two-dimensional nuclear magnetic resonance data. Compound 2 is a newly discovered C 1 -diterpenoid alkaloid, which was named Yili delphinidine B (Sinchianidine B). The 13 H and 1 C NMR data assignments are shown in Table 1 [600 MHz ( 13 H), 150 MHz (

[0036] Compound 3 is Yili delphinidine C, a colorless amorphous powder. Its molecular formula was determined to be C + H 24 H 35 NO8 by HRESI(+)MS (m / z 466.2436 [M+H] 1 , theoretical value 466.2435); its structure was determined based on 13 H, 19 C and two-dimensional nuclear magnetic resonance data. Compound 3 is a newly discovered C 1 -diterpenoid alkaloid, which was named Yili delphinidine C (Sinchianidine C). The 13 H and 1 C NMR data assignments are shown in Table 1 [600 MHz ( 13C), Solvent: CD3OD];

[0037] Compound 4 is ilicinine D, a colorless amorphous powder. Its molecular formula was determined to be C + by HRESI(+)MS (m / z 466.2437 [M+H] 24 ), with a theoretical value of 466.2435; 35 According to the 1 H, 13 C, and two-dimensional nuclear magnetic resonance data, its structure was determined. Compound 4 is a newly discovered C 19 -diterpenoid alkaloid, which was named ilicinine D (Sinchianidine D). The 1 H and 13 C NMR data assignments are shown in Table 1 [600 MHz ( 1 H), 150 MHz ( 13 C), Solvent: CD3OD].

[0038] Table 1 1 H (600 MHz) and 13 C (150 MHz) NMR data of Compounds 1 - 4 [δ (ppm), J (Hz)]

[0039]

[0040] BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the 1 H NMR spectrum of the compound ilicinine A of the present invention;

[0042] Figure 2 is the 13 C NMR spectrum of the compound ilicinine A of the present invention;

[0043] Figure 3 is the 1 H NMR spectrum of the compound ilicinine B of the present invention;

[0044] Figure 4 is the 13 C NMR spectrum of the compound ilicinine B of the present invention;

[0045] Figure 5 is the 1 H NMR spectrum of the compound ilicinine C of the present invention;

[0046] Figure 6 is the 13 C NMR spectrum of the compound ilicinine C of the present invention;

[0047] Figure 7 For the compound delsoline D of the present invention 1 H NMR spectrum;

[0048] Figure 8 For the compound delsoline D of the present invention 13 C NMR spectrum. Detailed implementation mode

[0049] 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, on-line 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 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);

[0050] Delphinium iliense, collected from the northern slope of Tianshan Mountains at an altitude of 1850 ± 20 m in Chahanwusu Township, Zhaosu County, Yili in July 2017. It was identified as Delphinium iliense Huth of the genus Delphinium in the family Ranunculaceae by Associate Researcher Feng Ying of Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences. The specimen is preserved in Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences.

[0051] Example 1 (two separations)

[0052] a. Take 10.0 kg of the whole herb of Delphinium iliense, crush it, soak the medicinal material thoroughly with 5% Na2CO3 solution at room temperature for 3 hours, add 50 L of a chloroform:methanol mixed solution with a volume ratio of 6:1, extract by cold maceration method, and evaporate the solvent under reduced pressure to obtain the crude extract paste of Delphinium iliense;

[0053] b. Disperse the crude extract paste obtained in step a with 1% sulfuric acid solution, separate the acid aqueous layer, adjust the pH to 10 with anhydrous Na2CO3, then extract with ethyl acetate, evaporate the ethyl acetate under reduced pressure to obtain the total alkaloid paste 1; then alkalize the alkaline aqueous layer with 10% NaOH to pH 12, extract with n-butanol, evaporate the n-butanol under reduced pressure to obtain the total alkaloid paste 2, and combine the total alkaloid paste 1 and total alkaloid paste 2 to obtain the total alkaloid paste;

[0054] c. Separate the obtained total alkaloid paste 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 10 components A-J;

[0055] 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 - 100:0, combine the same fractions after TLC and HPLC analysis to obtain 5 components A-1 to A-5; purify component A-2 by semi-preparative high-performance liquid chromatography, perform gradient elution with an acetonitrile - 0.1% formic acid water with a volume ratio of 20:80 - 35:65 to obtain the compound delsemine A;

[0056] Continue to further separate the obtained component C by normal-phase silica gel column chromatography, perform gradient elution with a 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 TLC and HPLC analysis to obtain 5 components C-1 to C-5; purify component C-2 by semi-preparative high-performance liquid chromatography, perform gradient elution with an acetonitrile - 0.1% formic acid water with a volume ratio of 20:80 - 35:65 to obtain the compound delsemine B;

[0057] The obtained fraction E was further separated by normal-phase silica gel column chromatography, and gradient elution was performed 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 TLC and HPLC, the same fractions were combined to obtain 4 fractions E-1 to E-4; fraction E-2 was purified by semi-preparative high-performance liquid chromatography with a gradient elution of acetonitrile-0.1% formic acid water with a volume ratio of 20:80 - 40:60 to obtain the compound iliciline C;

[0058] The obtained fraction I was further separated by normal-phase silica gel column chromatography, and gradient elution was performed using petroleum ether-acetone + 0.2% diethylamine with volume ratios of 10:1, 10:1, 5:1, 2:1, 1:1, and 0:1 as the eluent. After analysis by TLC and HPLC, the same fractions were combined to obtain 4 fractions I-1 to I-4; fraction I-4 was purified by semi-preparative high-performance liquid chromatography with a gradient elution of acetonitrile-0.1% formic acid water with a volume ratio of 20:80 - 40:60 to obtain the compound iliciline D;

[0059] The compounds iliciline A, iliciline B, iliciline C, and iliciline D obtained in step c were analyzed by thin-layer chromatography, developed with a developing agent of chloroform-methanol-water with a volume ratio of 82:16:2, to determine 4 new diterpenoid alkaloid monomer compounds.

[0060] Example 2 (Two Separations)

[0061] a. After crushing 10.0 kg of the whole herb of Delphinium iliense, it was extracted by percolation with 50 L of ethanol with a volume fraction of 80% at room temperature, and the solvent was evaporated under reduced pressure to obtain the crude extract paste of Delphinium iliense;

[0062] b. The crude extract paste obtained in step a was dispersed with sulfuric acid with a mass fraction of 2%, and 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;

[0063] c. The obtained total alkaloid extract paste was separated by Sephadex LH-20 gel column chromatography, and isocratic elution was performed using a volume ratio of 1:1 chloroform and methanol as the eluent. One fraction was collected every 20 ml, and the fractions were analyzed by silica gel thin-layer chromatography (TLC), and the same fractions were combined to obtain 8 fractions A - H;

[0064] The obtained fraction A was further separated by Sephadex LH-20 gel column chromatography, and gradient elution was carried out with methanol-water with a volume ratio of 5:95 - 80:20 as the eluent. After analysis by TLC and high performance liquid chromatography (HPLC), the same fractions were combined to obtain 6 fractions A-1 to A-6; fraction A-2 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, and compound delavaconitine A was obtained;

[0065] The obtained fraction C was further separated by Sephadex LH-20 gel column chromatography, and isocratic elution with methanol was carried out. After analysis by TLC and HPLC, the same fractions were combined to obtain 4 fractions C-1 to C-4; fraction C-3 was further separated and purified by preparative thin layer chromatography, developed with petroleum ether-acetone saturated with ammonia water with a volume ratio of 3:1 as the developing agent, and compound delavaconitine B was obtained;

[0066] The obtained fraction D was further separated by Sephadex LH-20 gel column chromatography, and gradient elution was carried out with methanol-water with a volume ratio of 5:95 - 80:20 as the eluent, and compound delavaconitine C was obtained;

[0067] The obtained fraction H was further separated by Sephadex LH-20 gel column chromatography, and gradient elution was carried out with methanol-water with a volume ratio of 5:95 - 80:20 as the eluent. After analysis by TLC and HPLC, the same fractions were combined to obtain 4 fractions H-1 to H-4; fraction H-3 was further separated and purified by preparative thin layer chromatography, developed with petroleum ether-acetone saturated with ammonia water with a volume ratio of 2:1 as the developing agent, and compound delavaconitine D was obtained;

[0068] The compounds delavaconitine A, delavaconitine B, delavaconitine C and delavaconitine D obtained in step c were detected and analyzed by high performance liquid chromatography, and gradient elution was carried out with acetonitrile-formic acid aqueous solution with a volume ratio of 10:90 - 80:20 to determine 4 new diterpenoid alkaloid monomer compounds.

[0069] Example 3 (Three Separations)

[0070] a. After 10.0 kg of the whole herb of Delphinium iliense was crushed, it was ultrasonically extracted with methanol, and the methanol was recovered by reduced pressure concentration to obtain the total extract;

[0071] b. Disperse the total extract obtained in step a in an aqueous hydrochloric acid solution with a mass fraction of 3%, filter to obtain the acidic aqueous solution, adjust the pH to 10 with anhydrous Na2CO3, then extract with ethyl acetate, and evaporate the ethyl acetate under reduced pressure to obtain the total alkaloid extract 1; further alkalize the alkaline aqueous 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 extract 2. Combine the total alkaloid extract 1 and the total alkaloid extract 2 to obtain the total alkaloid extract;

[0072] c. Separate the total alkaloid extract obtained in step b by normal-phase silica gel column chromatography, and perform gradient elution successively with chloroform-methanol 60:1, 40:1, 20:1, 10:1, 5:1, and 1:1 as the solvent systems. Collect one fraction every 500 ml. Analyze the fractions by silica gel thin-layer chromatography (TLC), and combine the same fractions to obtain 8 fractions A - H;

[0073] Further separate fraction A by reverse-phase silica gel (ODS) column chromatography, and perform gradient elution with a methanol-aqueous solution with a volume ratio of 5:95 - 80:20. After analysis by silica gel thin-layer chromatography (TLC) and HPLC, combine the same fractions to obtain 5 fractions A - 1 to A - 5; Purify fraction A - 2 by semi-preparative high-performance liquid chromatography, and perform gradient elution with an acetonitrile - 0.1% formic acid gradient with a volume ratio of 10:90 - 40:60 to obtain compounds delavine A and delavine B;

[0074] Separate fraction C by Sephadex LH-20 gel column chromatography, and perform gradient elution with a methanol-water with a volume ratio of 5:95 - 80:20 as the eluent. After analysis by TLC and HPLC, combine the same fractions to obtain 4 fractions C - 1 to C - 4; Purify C - 2 by semi-preparative high-performance liquid chromatography, and perform gradient elution with an acetonitrile - 0.1% formic acid gradient with a volume ratio of 22:78 - 35:65 to obtain compound delavine C;

[0075] Separate fraction E by Sephadex LH-20 gel column chromatography, and perform gradient elution with a methanol-water with a volume ratio of 5:95 - 80:20 as the eluent. After analysis by TLC and HPLC, combine the same fractions to obtain 3 fractions E - 1 to E - 3; Purify fraction E - 2 by semi-preparative high-performance liquid chromatography, and perform gradient elution with an acetonitrile - 0.1% formic acid gradient with a volume ratio of 20:80 - 35:65 to obtain compound delavine D;

[0076] Analyze the compounds delavine A, delavine B, delavine C, and delavine D obtained in step c by thin-layer chromatography, and develop with a petroleum ether-acetone saturated with ammonia water with a volume ratio of 3:1 as the developing agent to determine 4 new diterpenoid alkaloid monomer compounds.

[0077] Example 4 (Three Separations)

[0078] a. Take 10.0 kg of the whole herb of Delphinium iliense, crush it, and ultrasonically extract it with 50 L of chloroform at room temperature. Evaporate the solvent under reduced pressure to obtain the crude extract paste of Delphinium iliense;

[0079] b. Disperse the crude extract paste obtained in step a with hydrochloric acid with a mass fraction of 4% to obtain an acidic aqueous solution. Adjust the pH to 10 with ammonia water, and then extract with ethyl acetate. Evaporate the ethyl acetate under reduced pressure to obtain the total alkaloid extract 1. Then, continue to alkalize the alkaline aqueous layer with 10% NaOH to a pH value of 12, extract with n-butanol, and evaporate the n-butanol under reduced pressure to obtain the total alkaloid extract 2. Combine the total alkaloid extract 1 and the total alkaloid extract 2 to obtain the total alkaloid extract;

[0080] c. Subject the total alkaloid extract obtained in step b to reverse-phase silica gel (ODS) column chromatography separation, and elute it with a methanol-aqueous solution gradient with a volume ratio of 5:95 - 100:0. 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;

[0081] Separate fraction A by Sephadex LH-20 gel column chromatography, and elute it with a methanol-water gradient with a volume ratio of 5:95 - 80:20 as the eluent. Combine the same fractions after TLC and HPLC analysis to obtain 4 fractions A-1 to A-4. Purify fraction A-3 by preparative thin-layer chromatography, and develop it with a petroleum ether-acetone saturated with ammonia water with a volume ratio of 3:1 to obtain the compound delphinidine A;

[0082] Separate the obtained fraction B by Sephadex LH-20 gel column chromatography, and elute it with a methanol-water gradient with a volume ratio of 5:95 - 80:20 as the eluent. Combine the same fractions after TLC and HPLC analysis to obtain 4 fractions B-1 to B-4. Purify fraction B-2 by preparative thin-layer chromatography, and develop it with a chloroform-methanol-water with a volume ratio of 82:16:2 as the developing agent to obtain the compound delphinidine B;

[0083] Separate the obtained fraction D by Sephadex LH-20 gel column chromatography, and elute it with a methanol-water gradient with a volume ratio of 5:95 - 80:20 as the eluent to obtain delphinidine C;

[0084] Separate the obtained fraction E by normal-phase silica gel column chromatography, and elute it successively with chloroform-methanol 40:1, 20:1, 10:1, 5:1, and 1:1 as the solvent system for gradient elution to obtain the compound delphinidine D;

[0085] The delavindine A, delavindine B, delavindine C, and delavindine D obtained in step c were analyzed by high performance liquid chromatography (HPLC), and gradient elution was performed with an acetonitrile-formic acid aqueous solution with a volume ratio of 25:75 - 80:20 to determine 4 new diterpenoid alkaloid monomer compounds.

[0086] Example 5 (Four Separations)

[0087] a. After crushing 10.0 kg of the whole plant of Delphinium iliense, it was refluxed and extracted with 50 L of 95% ethanol by volume at room temperature, and the solvent was evaporated under reduced pressure to obtain an extractive paste of the crude extract of Delphinium iliense.

[0088] b. The extractive paste of the crude extract obtained in step a was dispersed with 2% sulfuric acid by mass to obtain an acidic aqueous solution. The pH value was adjusted to 12 with 5% NaOH, and it was extracted with n-butanol. The n-butanol was evaporated under reduced pressure to obtain an extractive paste of total alkaloids.

[0089] c. The obtained extractive paste of total alkaloids was separated by Sephadex LH-20 gel column chromatography, and isocratic elution was performed with a 1:1 volume ratio of dichloromethane and methanol 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.

[0090] Component A was further separated by reverse phase silica gel (ODS) column method, and gradient elution was performed with a methanol-aqueous solution with a volume ratio of 5:95 - 80:20. After TLC and HPLC analysis, the same fractions were combined to obtain 4 components B-1 to B-4. Component B-2 was purified by preparative thin layer chromatography, developed with a 10:1 volume ratio of chloroform-methanol as the developing agent, and compound delavindine A was obtained.

[0091] The obtained component C was further separated by normal phase silica gel column chromatography, and gradient elution was performed with a volume ratio of 20:1, 10:1, 5:1, 2:1, 1:1, and 0:1 of petroleum ether-acetone + 0.2% diethylamine as the eluent. After TLC and HPLC analysis, 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, and gradient elution was performed with a volume ratio of 22:78 - 35:65 of acetonitrile-0.1% formic acid to obtain compound delavindine B.

[0092] The obtained fraction D was separated by Sephadex LH-20 gel column chromatography, and isocratic elution was carried out with a 1:1 (v / v) mixture of dichloromethane and methanol as the eluent. One fraction was collected every 10 ml. The fractions were analyzed by silica gel thin layer chromatography (TLC), and the same fractions were combined to obtain six fractions D-1 to D-6; fraction D-2 was purified by semi-preparative high performance liquid chromatography with a gradient elution of acetonitrile-0.1% formic acid in a volume ratio of 22:78 - 35:65 to obtain the compound iliciline C;

[0093] The obtained fraction E was further separated by normal phase silica gel column chromatography, and gradient elution was carried out with petroleum ether-acetone + 0.2% diethylamine in volume ratios of 10:1, 10:1, 5:1, 2:1, 1:1, and 0:1 as the eluent. After TLC and HPLC analysis, the same fractions were combined to obtain four fractions E-1 to E-4; fraction E-3 was purified by semi-preparative high performance liquid chromatography with a gradient elution of acetonitrile-0.1% formic acid in a volume ratio of 22:78 - 35:65 to obtain the compound iliciline D;

[0094] The compounds iliciline A, iliciline B, iliciline C, and iliciline D obtained in step c were analyzed by thin layer chromatography, developed with a developing agent of chloroform-methanol-water in a volume ratio of 82:16:2 to determine four new diterpenoid alkaloid monomer compounds.

[0095] Example 6 (Four Separations)

[0096] a. After 10.0 kg of the whole herb of Delphinium iliense was crushed, it was ultrasonically extracted with 50 L of methanol at room temperature, and the solvent was evaporated under reduced pressure to obtain an extract of the crude extract of Delphinium iliense;

[0097] b. The extract of the crude extract obtained in step a was dispersed with sulfuric acid with a mass fraction of 3% to obtain an acidic aqueous solution. The pH value was adjusted to 11 with 5% NaOH, and it was extracted with n-butanol. The n-butanol was evaporated under reduced pressure to obtain an extract of total alkaloids;

[0098] c. The extract of total alkaloids obtained in step b was separated by normal phase silica gel column chromatography, and gradient elution was carried out successively with chloroform-methanol in 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 five fractions A - E;

[0099] The obtained component A was further separated by reverse-phase silica gel (ODS) column chromatography and eluted with a gradient of methanol-aqueous solution with a volume ratio of 5:95 - 100:0. 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 water with a volume ratio of 10:90 - 40:60 to obtain compound delsemine A;

[0100] The obtained component C was separated by Sephadex LH-20 gel column chromatography and eluted with a gradient using methanol-water with a volume ratio of 5:95 - 80:20 as the eluent. After analysis by TLC and 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 compound delsemine B;

[0101] The obtained component D was further separated by normal-phase silica gel column chromatography and eluted with a gradient using petroleum ether-acetone + 0.2% diethylamine with a volume ratio of 10:1, 10:1, 5:1, 2:1, 1:1, and 0:1 as the eluent to obtain compound delsemine C;

[0102] The obtained component E was further separated by normal-phase silica gel column chromatography and eluted with a gradient using petroleum ether-acetone + 0.2% diethylamine with a volume ratio of 10:1, 10:1, 5:1, 2:1, 1:1, and 0:1 as the eluent to obtain 5 components E-1 to E-4; Component E-2 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 - 30:70 to obtain compound delsemine D;

[0103] The compounds delsemine A, delsemine B, delsemine C, and delsemine D obtained in step c were analyzed by thin-layer chromatography and developed with a developing agent of chloroform-methanol-water with a volume ratio of 82:16:2 to determine 4 new monomeric diterpenoid alkaloid compounds.

[0104] Example 7

[0105] The use of the diterpenoid alkaloid compounds isolated from Delphinium iliense Huth in the preparation of analgesic drugs, taking ICR mice as an example:

[0106] Screening for the analgesic activity of the obtained diterpenoid alkaloid compounds:

[0107] Experimental animals:

[0108] ICR mice, weighing 18 - 22 g, with an equal number of males and females, were provided by the Experimental Animal Department of Xinjiang Medical University. The mice were housed in an SPF - level animal experiment center at an indoor temperature of 22 ± 2 °C and a relative humidity of 40% - 70%, with free access to water and food.

[0109] Experimental instruments and reagents:

[0110] Dimethyl sulfoxide (DMSO), glacial acetic acid (Aladdin), sodium chloride injection (Xinjiang Huashidan Pharmaceutical Co., Ltd.);

[0111] Experimental content:

[0112] Preparation of drug solutions: The drugs were dissolved in dimethyl sulfoxide (DMSO) or 0.1 M / L hydrochloric acid, and then diluted with physiological saline. The blank control group was the solvent control, and they were stored in the dark at room temperature.

[0113] Preparation of 0.6% acetic acid solution: 10 ml of 10% aqueous acetic acid solution was prepared 30 min before preparing the model and placed in an ice - water box. 25 min later, 360 μl of 10% acetic acid was taken to prepare 6 ml of 0.6% acetic acid solution (enough for 10 mice) and placed in the ice - water box. It was used immediately after preparation.

[0114] Acetic acid writhing test in mice: 50 ICR mice, with an equal number of males and females, were randomly divided into 5 groups: a blank control group and each test drug group, with 10 mice in each group. Each group of mice was intraperitoneally injected with a drug at a volume of 0.1 mL / 10 g, and the blank control group was given the blank solvent system. 30 min after drug administration to each group of mice, 0.1 mL / 10 g of 0.6% glacial acetic acid was intraperitoneally injected, and the number of writhing times of the mice within 20 min was recorded. The pain inhibition percentage of each drug - administered 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 - administered group) / Number of writhing times in the blank control group × 100%. The experimental results are shown in Table 2.

[0115] Table 2 Effects of the new diterpenoid alkaloid compounds delsemine C and delsemine D in Delphinium iliense Huth on acetic - acid - induced pain in mice

[0116]

[0117] To study the analgesic effects of the new diterpenoid alkaloid compounds delsemine C and delsemine D isolated from Delphinium iliense Huth on acetic - acid - induced abdominal pain, this experiment established a pain model by intraperitoneally injecting acetic acid into mice, and observed the effects of compounds delsemine C and 4 on the number of writhing times and writhing latency of mice, so as to evaluate their curative effects.

[0118] The experimental results show that: the new diterpenoid alkaloid compounds delsemine C and delsemine D isolated from Delphinium iliense Huth described in the present invention significantly reduce the writhing times of pain-induced mice and prolong their writhing latency. Therefore, it can be concluded that the new diterpenoid alkaloid compounds delsemine C and delsemine D isolated from Delphinium iliense Huth have an obvious analgesic effect on acetic acid-induced abdominal pain, and the analgesic effect of compound delsemine C shows a dose-effect relationship. In summary, the above results provide an experimental basis for whether this compound can be used as a potential clinical analgesic.

Claims

1. A diterpene alkaloid compound isolated from Delphinium iliense flower, characterized in that The structural formula of the compound is: in: The name of compound 1 is Ili delphinidin A; The name of compound 2 is Ili delphinidin B; The name of compound 3 is Ili delphinidin C; The name of compound 4 is delphinium ili D.

2. The method for preparing diterpene alkaloid compounds from Delphinium iliense flowers according to claim 1, characterized in that Follow these steps: a. After the raw material, the whole herb of Ili Delphinium is crushed, an aqueous ethanol solution with a volume fraction of 10-95%, anhydrous methanol or chloroform is used at room temperature, and anhydrous methanol and chloroform are extracted by cold soaking, percolation, heating reflux or ultrasonic extraction, 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 aqueous solution 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 or n-butanol, and concentrated under reduced pressure to recover the organic solvent to obtain total alkaloids; Or the crude extract obtained in step a is dispersed with sulfuric acid or hydrochloric acid with a mass fraction of 1-5%, and the obtained acid aqueous solution is adjusted to pH=10 with ammonia water and anhydrous Na2CO3, and then extracted with ethyl acetate, and the ethyl acetate is evaporated under reduced pressure to obtain total alkaloid extract 1; the alkaline water layer is further alkalized with 10% NaOH to a pH value of 12, and then extracted with n-butanol, and the n-butanol is evaporated under reduced pressure to obtain total alkaloid extract 2, and the total alkaloid extract 1 and the total alkaloid extract 2 are combined to obtain the total alkaloid extract; c. Separating the total alkaloids obtained in step b by two, three or four methods including silica gel column chromatography, thin layer chromatography, dextran gel LH-20 column chromatography or high performance liquid chromatography to obtain compound 1 delphinidin A, compound 2 delphinidin B, compound 3 delphinidin C and compound 4 delphinidin D. The crude extract obtained in step a is dispersed with hydrochloric acid having a mass fraction of 4%, and the obtained acid aqueous solution is adjusted to pH=10 with ammonia water, and then extracted with ethyl acetate, and the ethyl acetate is evaporated under reduced pressure to obtain total alkaloid extract 1; the alkaline water layer is further alkalized with 10% NaOH to a pH value of 12, and then extracted with n-butanol, and the n-butanol is evaporated under reduced pressure to obtain total alkaloid extract 2, and the total alkaloid extract 1 and the total alkaloid extract 2 are combined to obtain the total alkaloid extract.

3. The method for preparing diterpene alkaloid compounds from Delphinium iliense flowers according to claim 2, characterized in that Two separations in step c: The total alkaloids obtained in step c are separated by normal phase silica gel column chromatography, and gradient elution is performed using dichloromethane 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, and 1:1 as eluents, and a fraction is collected every 500 ml. The fractions are analyzed by silica gel thin layer chromatography, and the same fractions are combined to obtain 8 components AH; 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 TLC and HPLC analysis, the same fractions are combined to obtain five components A-1 to A-5; component A-3 is purified by semi-preparative high performance liquid chromatography and isocratically eluted with acetonitrile-0.1% formic acid aqueous solution in a volume ratio of 29:71 to obtain compounds delphinidin A and delphinidin B; Component C among the eight components AH obtained 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 HPLC analysis, the same fractions are combined to obtain six components C-1 to C-6; component C-3 is purified by semi-preparative high performance liquid chromatography, and gradient elution is performed using acetonitrile-0.1% formic acid aqueous solution in a volume ratio of 20:80-30:70 to obtain compounds delphinidin C and delphinidin D.

4. The method for preparing diterpene alkaloid compounds from Delphinium iliense flowers according to claim 2, characterized in that Three separations in step c: The total alkaloid extract obtained in step c is separated by normal phase silica gel column chromatography or reverse phase silica gel column chromatography, and gradient elution is performed 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, and a fraction is collected every 500 ml. After analysis by silica gel thin layer chromatography, the same fractions are combined to obtain 6 components AF; 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 in a volume ratio of 5:95-70:30 or petroleum ether-acetone + 0.2% diethylamine in a volume ratio of 10:1, 5:1, 2:1, 1:1 and 0:1 as an eluent, and the same fractions are combined after silica gel thin layer chromatography and HPLC analysis to obtain five components A-1 to A-5; component A-3 is purified by semi-preparative high performance liquid chromatography, and then gradient eluted with acetonitrile-0.1% formic acid aqueous solution in a volume ratio of 20:80-40:60 to obtain compounds delphinidin A and delphinidin B; The obtained component C was further separated by dextran LH-20 gel column chromatography, and isocratically eluted with chloroform-methanol in a volume ratio of 1:1 or 0:

1. After silica gel thin layer chromatography and HPLC analysis, the same fractions were combined to obtain 5 components C-1 to C-5; C-4 was purified by semi-preparative high performance liquid chromatography, and isocratically eluted with acetonitrile-0.1% formic acid in a volume ratio of 28:72 to obtain compounds delphinidin C and delphinidin D.

5. The method for preparing diterpene alkaloid compounds from Delphinium iliense flowers according to claim 2, characterized in that Four separations in step c: The total alkaloid extract obtained in step c is separated by Sephadex LH-20 column chromatography, and isocratically eluted using a chloroform-methanol solvent system with a volume ratio of 1:1 or 0:1, and the fractions are analyzed by silica gel thin layer chromatography, and the same fractions are combined to obtain 9 fractions of component AI; 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 delphinidin A. The obtained component C was subjected to normal phase silica gel column chromatography, and gradient elution was 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 as eluents. After silica gel thin layer chromatography and HPLC 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, and chloroform-methanol-water in a volume ratio of 82:16:2 was used as a developing solvent to obtain compound delphinidin B; The obtained component E was subjected to normal phase silica gel column chromatography, and gradient elution was performed using petroleum ether-acetone + 0.2% diethylamine in a volume ratio of 10:1, 5:1, 2:1, 1:1 and 0:1 as eluents. After silica gel thin layer chromatography and HPLC analysis, the same fractions were combined to obtain 5 components E-1 to E-5; component E-3 was purified by semi-preparative high performance liquid chromatography, and gradient elution was performed using acetonitrile-0.1% formic acid aqueous solution in a volume ratio of 20:80-47:53 to obtain compound delphinidin C; The obtained component I was separated by Sephadex LH-20 column chromatography, and isocratically eluted with chloroform-methanol as a solvent system with a volume ratio of 1:1 or 0:

1. The fractions were analyzed by silica gel thin layer chromatography, and the same fractions were combined to obtain 4 segments of components I-1 to I-4; component I-4 was purified by semi-preparative high performance liquid chromatography, and isocratically eluted with acetonitrile-0.1% formic acid aqueous solution with a volume ratio of 28:72 to obtain compound delphinidin D.

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

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