Alkaloid compound as well as preparation method and application thereof
By combining multiple column chromatography techniques, six new alkaloid compounds were isolated from the whole herb of Viola tianshanensis, solving the problem of the lack of analgesic active substance basis in existing technologies and realizing the effective separation and analgesic activity identification of the compounds.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- XINJIANG TECH INST OF PHYSICS & CHEM CHINESE ACAD OF SCI
- Filing Date
- 2026-02-10
- Publication Date
- 2026-05-19
AI Technical Summary
In the current technology, the extraction, separation and pharmacological study of alkaloid components in Viola tianshanensis are still in the initial stage, lacking a clear basis for analgesic active substances and highly effective and low-toxicity drug lead compounds.
Six new alkaloid compounds, including violane AF, were isolated from the whole herb of Viola tianshanensis using a combination of solvent extraction and multiple column chromatography techniques. The compounds were purified by gradient elution using silica gel column chromatography, thin-layer chromatography, polyamide column chromatography, macroporous resin column chromatography, dextran gel LH-20 column chromatography, and high-performance liquid chromatography.
Six new alkaloid compounds were successfully isolated and identified, showing moderate to strong analgesic activity and potential value for analgesic drug applications.
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Figure CN122059927A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical technology, and in particular to an alkaloid compound, its preparation method, and its uses. Background Technology
[0002] Alkaloids are a class of nitrogen-containing basic organic compounds found in nature (mainly in plants, but some also in animals). Most have complex ring structures, with nitrogen largely contained within the ring. They possess significant biological activity and are among the important active ingredients in traditional Chinese medicine. Clinical applications include the treatment of tumors and pain, as well as diabetes and Alzheimer's disease.
[0003] Viola kunawurensis Royle Illustr., belonging to the Violaceae family and the Viola genus, is a perennial herb that grows in alpine or subalpine meadows and on mountain slopes. It is distributed in the Tianshan Mountains, Kunlun Mountains, and Pamir Plateau of Xinjiang, as well as Yunnan, Sichuan, and Tibet in my country, and is also found in Central Asia. The entire plant is used medicinally and is a commonly used ingredient in traditional Uyghur medicine (commonly known as Binafuxi). It is used as the main ingredient in many Uyghur medicine preparations, such as Viola kunawurensis pills, compound Viola kunawurensis granules, Aibibinafuxie pills, and Hemiribinafuxie syrup.
[0004] In China, the analgesic activity of alkaloids has been extensively studied. Currently, morphine hydrochloride combined with atropine is effective in treating severe, persistent pain in patients with end-stage malignant intestinal obstruction. Repeated use during pain recurrence reduces drowsiness, and no serious adverse reactions have been observed. Strychnos nux-vomica aqueous extract contains strychnine, an active ingredient that can alleviate pain, swelling, and bone destruction caused by rheumatoid arthritis. Currently, the extraction, isolation, structural identification, and related pharmacological research of alkaloids from Violets, especially Viola tianshanense, are still in their initial stages both domestically and internationally. Therefore, systematic and in-depth research on the alkaloids in Viola tianshanense to clarify the material basis of their analgesic activity, discover new alkaloid compounds with specific activity, and identify highly effective and low-toxicity drug lead compounds is of great significance. Summary of the Invention
[0005] The purpose of this invention is to provide an alkaloid compound, its preparation method, and its uses. The compound is obtained from the whole herb of *Viola tianshanense*, after alkalization treatment, by solvent extraction and separation using two, three, or four of the following methods: silica gel column chromatography, thin-layer chromatography, polyamide column chromatography, macroporous resin column chromatography, dextran gel LH-20 column chromatography, and high-performance liquid chromatography, yielding six new alkaloids: compound 1 is named *Viola tianshanense* alkaloid A, compound 2 is named *Viola tianshanense* alkaloid B, compound 3 is named *Viola tianshanense* alkaloid C, compound 4 is named *Viola tianshanense* alkaloid D, compound 5 is named *Viola tianshanense* alkaloid E, and compound 6 is named *Viola tianshanense* alkaloid F. The analgesic activity of the obtained compounds was determined. Experimental results showed that compound *Viola tianshanense* AF exhibited moderate to strong analgesic activity in an acetic acid-induced writhing mouse model, and can be used to prepare analgesic drugs.
[0006] To achieve the above objectives, the basic solution provided by this invention is: an alkaloid compound comprising the following compounds, the structural formulas of which are as follows: A method for preparing an alkaloid compound includes the following steps: a. After pulverizing the whole herb of Viola tianshanensis, mix it thoroughly with a mixture of ammonia and acetone at room temperature to obtain the soaked medicinal material; b. At room temperature, the medicinal material obtained in step a is extracted by cold soaking in a mixed solution of chloroform and methanol, and the organic solvent is recovered by vacuum concentration to obtain an extract; c. Dissolve the extract obtained in step b with 5 times its volume of water, then extract it 5 times with the same volume of ethyl acetate as water. Take the ethyl acetate layer sample, concentrate it under reduced pressure to recover the ethyl acetate, and obtain the total alkaloids. d. The total alkaloids obtained in step c are separated by two, three, or four of the following methods: silica gel column chromatography, thin-layer chromatography, polyamide column chromatography, macroporous resin column chromatography, dextran gel LH-20 column chromatography, and high-performance liquid chromatography. Furthermore, the two separation methods in step d are as follows: The total alkaloid extract obtained in step c was separated by silica gel column chromatography. Gradient elution was performed using petroleum ether and diethylamine in a volume ratio of 100:1 to 8:2. The eluted fractions were collected and analyzed by thin-layer chromatography. The same samples were combined to obtain 8 components, which were numbered 1-8 in sequence. Fraction 6 was further separated by silica gel column chromatography, using petroleum ether and acetone in volume ratios of 19:1 to 8:2 as eluents for gradient elution. The eluted samples were collected and combined after thin-layer chromatography analysis to obtain 7 subfractions, numbered 6A-6G. Fraction 6B was used to remove pigment impurities by dextran gel column chromatography with chloroform and methanol in a 1:1 volume ratio as eluent. Samples with lighter or colorless color were collected. The samples with removed pigment impurities were purified by semi-preparative high performance liquid chromatography with acetonitrile-water in a 30:70 volume ratio as mobile phase. Peaks with retention times of 18 min and 25 min were collected and concentrated to obtain compounds tianshan violacein A and tianshan violacein B. Fraction 6E was separated by silica gel column chromatography, using petroleum ether and ethyl acetate in a volume ratio of 19:1 to 8:2 as eluents for gradient elution. The eluted samples were collected and analyzed by thin-layer chromatography. The same samples were combined to obtain four subfractions, numbered 6Ea-6Ed. Fraction 6Ea was purified by semi-preparative high-performance liquid chromatography (HPLC) with acetonitrile-water in a volume ratio of 23:77. The peak with a retention time of 21 min was collected and concentrated to obtain the compound colophonine C. The fraction 6F was separated by polyamide column chromatography and eluted isocratically with 30% methanol aqueous solution. The eluted sample was collected and concentrated. The sample was purified by semi-preparative high performance liquid chromatography to remove pigment impurities. The mobile phase was acetonitrile-water with a volume ratio of 15:85. The peaks with retention times of 15 min and 22 min were collected and concentrated to obtain compounds tianshan violacein D and tianshan violacein E. Fraction 6G was used to remove pigment impurities by dextran gel column chromatography with methanol as the eluent. Samples with lighter or colorless color were collected. Fraction 6G after removing pigment impurities was then purified by semi-preparative high performance liquid chromatography with acetonitrile-water at a volume ratio of 18:82 as the mobile phase. The peak with a retention time of 31 min was collected and concentrated to obtain the compound colophonine F.
[0007] Furthermore, the three separation methods in step d are as follows: The total alkaloid extract obtained in step c was separated by silica gel column chromatography. Gradient elution was performed using petroleum ether and acetone in a volume ratio of 100:1 to 8:2. The eluted fractions were collected and analyzed by thin-layer chromatography. The same samples were combined to obtain 8 components, which were numbered 1-8 in sequence. Fraction 4 was further separated by silica gel column chromatography, using chloroform and methanol in volume ratios of 19:1, 9:1, 17:3, and 8:2 as eluents for gradient elution. The eluted samples were collected, and after thin-layer chromatography analysis, the same samples were combined to obtain four subfractions, numbered 4A-4D. Fraction 4A was separated by polyamide column chromatography and eluted isocratically with a 20% (v / v) methanol aqueous solution. The eluted sample was collected and concentrated, and then dextran gel column chromatography was used to remove pigment impurities. Methanol was used as the eluent. Samples with lighter color or no color were collected. The sample with removed pigment impurities was purified by semi-preparative high performance liquid chromatography with acetonitrile-water at a v / v ratio of 25:75. Peaks with retention times of 25 min and 33 min were collected, respectively. After concentration, compounds tianshan violacein A and tianshan violacein B were obtained. Fraction 4B was separated by polyamide column chromatography using a gradient elution with petroleum ether and acetone in volume ratios of 19:1, 9:1, 17:3, and 8:2. The eluted samples were collected and analyzed by thin-layer chromatography. Samples of the same type were then combined to obtain three subfractions, numbered 4Ba-4Bc. Fraction 4Ba was purified by semi-preparative high-performance liquid chromatography (HPLC) using acetonitrile-water in a mobile phase of 20:80. The peak with a retention time of 25 min was collected and concentrated to obtain the compound colophonine C. Fraction 4C was separated by macroporous resin column chromatography, eluted with a gradient of 30%-80% methanol-water solution. The eluted sample was collected and concentrated, and the fractions were combined by analytical high-performance liquid chromatography to obtain four subfractions, numbered 4Ca-4Cd. Pigment impurities in fraction 4Cb were removed by dextran gel column chromatography, with methanol and acetone in a 1:1 volume ratio as the eluent. Samples with lighter or colorless colors were collected. The sample after removing pigment impurities was purified by semi-preparative high-performance liquid chromatography, with acetonitrile-water in a 18:82 volume ratio as the mobile phase. Peaks with retention times of 18 min and 26 min were collected, and the concentrations yielded compounds tianshan violacein D and tianshan violacein E. The 4D fraction was purified by dextran gel column chromatography to remove pigment impurities using methanol as the eluent. Lighter or colorless samples were collected and then purified by macroporous resin column chromatography using 15% ethanol-water as the eluent. The eluted samples were collected in fractions and concentrated to obtain the compound colophonine F and subfraction 4Da.
[0008] Furthermore, the four separation methods in step d are as follows: The total alkaloid extract obtained in step c was separated by macroporous resin column chromatography. Gradient elution was performed using ethanol and water with volume fractions of 10% to 100% as eluents. The eluted fractions were collected, and after thin-layer chromatography analysis, the same samples were combined to obtain 5 components, numbered 1-5 in sequence. Fraction 3 was separated by polyamide column chromatography, and gradient elution was performed using chloroform and methanol in volume ratios of 19:1 to 8:2. The eluted samples were collected, and after thin-layer chromatography analysis, the same samples were combined to obtain 4 subfractions, numbered 3A-3D. Separation fraction 3A was performed by silica gel column chromatography, eluting sequentially with dichloromethane and acetone in a volume ratio of 19:1 to 8:2. The eluted samples were collected and concentrated, and then pigment impurities were removed by polyamide column chromatography with 20% methanol-water as the eluent. Samples with lighter or colorless colors were collected. The samples after removing pigment impurities were purified by semi-preparative high-performance liquid chromatography with acetonitrile-water in a volume ratio of 25:75. Peaks with retention times of 25 min and 33 min were collected, respectively. After concentration, compounds tianshan violacein A and tianshan violacein B were obtained. Fraction 3B was separated by macroporous resin column chromatography using 30% methanol-water as eluent for isocratic elution. The eluted samples were collected in fractions, and after thin-layer chromatography analysis, identical samples were combined to obtain four subfractions, numbered 3Ba-3Bd. Fraction 3Bd was purified by dextran gel column chromatography using chloroform and acetone in a 1:1 volume ratio as eluent. The eluted samples were collected in fractions, concentrated, and yielded the compound corydaline C and subfractions 3Bd1 and 3Bd2. Fraction 3C was separated by dextran gel chromatography with methanol and acetone in a volume ratio of 2:1 as the eluent. The eluted sample was collected and concentrated, and then combined by thin-layer chromatography to obtain two subfractions, numbered 3Ca and 3Cb, respectively. 3Ca and 3Cb were purified by semi-preparative high-performance liquid chromatography with methanol-water in a volume ratio of 20:80 for both fractions. The peaks with retention times of 20 min and 28 min were collected, respectively, and then concentrated to obtain compounds tianshan violacein D and tianshan violacein E. Fraction 3D was separated by macroporous resin column chromatography, using a gradient elution with methanol-water at a volume fraction of 45%–100%. The eluted fractions were collected and analyzed by analytical high-performance liquid chromatography, and then combined into four subfractions, numbered 3Da-3Dd. Fraction 3Dc was separated by dextran gel chromatography, using chloroform and acetone in a 1:1 volume ratio as the eluent. The eluted samples were collected in fractions and concentrated to obtain the compound corydaline F and subfraction 3Dc1.
[0009] The use of an alkaloid compound in the preparation of analgesic drugs.
[0010] The compound 1 described in this invention is named Violatin A, and is a yellow needle crystal. It was analyzed by HRESI (+) MS (m / z 294.1814 [M+H]). + Its molecular formula is determined to be C (theoretical value 294.1739). 15 H 23 N3O3; according to 1 H, 13 Its structure was determined using C and two-dimensional NMR data, and it was named tianshan corydaline A. 1H and 13 The C NMR data attribution is shown in Table 1 [600 MHz ( 1 H), 150 MHz 13 C), Solvent: CD3OD]; Compound 2, named Violatinibone B, is a brown gelatinous substance. It was analyzed by HRESI (+) MS (m / z 335.1753 [M+H]). + (The theoretical value is 335.1681), its molecular formula is determined to be C. 21 H 22 N2O2; according to 1 H, 13 Its structure was determined using C and two-dimensional NMR data, and it was named colophonine B. 1 H and 13 The C NMR data attribution is shown in Table 1 [600 MHz ( 1 H), 150 MHz 13 C), Solvent: CD3OD]; Compound 3, named Violatinine C, is a pale yellow gelatinous substance. It was analyzed by HRESI (+) MS (m / z 261.1601 [M+H]). + (Theoretical value 261.1525) determines its molecular formula as C 15 H 20 N2O2; according to 1 H, 13 Its structure was determined using C and two-dimensional NMR data, and it was named tianshan corydaline C. 1 H and 13 The C NMR data attribution is shown in Table 1 [600 MHz ( 1 H), 150 MHz 13 C), Solvent: CD3OD]; Compound 4, named tianshan violacein D, is a white needle crystal. It was analyzed by HRESI (+) MS (m / z 266.1743 [M+H]). + Its molecular formula is determined to be C (theoretical value 266.1678). 15 H 23 NO3; According to 1 H, 13 Its structure was determined using C and two-dimensional NMR data, and it was named tianshan corydaline D. 1 H and 13 The C NMR data attribution is shown in Table 2 [600 MHz ( 1 H), 150 MHz 13 C), Solvent: CD3OD]; Compound 5, named Violatinine E, is a white, blocky crystal. It was analyzed by HRESI (+) MS (m / z 224.1281 [M+H]). + (The theoretical value is 224.1208), its molecular formula is determined to be C. 12 H 17 NO3; According to 1 H, 13 Its structure was determined by C and two-dimensional nuclear magnetic resonance data, and it was named tianshan violacein E. 1 H and 13 The C NMR data attribution is shown in Table 2 [600 MHz ( 1 H), 150 MHz 13 C), Solvent: CD3OD]; Compound 6, named Violatinine F, is a pale yellow gelatinous substance. It was analyzed by HRESI (+) MS (m / z 208.1327 [M+H)). + Its molecular formula is determined to be C (theoretical value 208.2730). 12 H 17 NO2; According to 1 H, 13 Its structure was determined by C and two-dimensional nuclear magnetic resonance data, and it was named colophonine F. 1 H and 13 The C NMR data attribution is shown in Table 2 [600 MHz ( 1 H), 150 MHz 13 C), Solvent: CD3OD]; Table 1. Compounds 1-3 1 H (600 MHz) and 13 C (150 MHz) NMR data [δ (ppm), J (Hz)] Table 2. Compounds 3-6 1 H (600 MHz) and 13 C (150 MHz) NMR data [δ (ppm), J (Hz)] Attached Figure Description
[0011] Figure 1 The 1H NMR spectrum of compound 1, Violane A, of this invention; Figure 2 The 13C NMR spectrum of compound 1, Violane A, of this invention; Figure 3 The 1H NMR spectrum of compound 2-tianshan violacein B of the present invention; Figure 4 The 13C NMR spectrum of compound 2, Violane B, of this invention; Figure 5 The 1H NMR spectrum of compound C3, tianshan violacein, is shown below. Figure 6 The 13C NMR spectrum of compound C, 3-tianshan violacein, is shown below. Figure 7 The 1H NMR spectrum of compound 4-tianshan violacein D of the present invention; Figure 8 The 13C NMR spectrum of compound 4-tianshan violacein D of the present invention; Figure 9 The 1H NMR spectrum of the compound 5-dayiviolacein E of this invention; Figure 10 The 13C NMR spectrum of the compound 5-dayiviolacein E of this invention; Figure 11 The 1H NMR spectrum of compound 6-tianshan violacein F of this invention; Figure 12 This is the 13C NMR spectrum of compound 6-tianshan violacein F of the present invention. Detailed Implementation
[0012] The present invention will be further described in detail below through specific embodiments: like Figures 1 to 12 As shown: All reagents used were of analytical grade. Acetonitrile used in high-performance liquid chromatography (HPLC) was HPLC grade (Merk, USA). Column chromatography silica gel (100-200 mesh, 200-300 mesh): produced by Qingdao Marine Chemical Plant; Sephadex LH-20 gel: produced by Pharmacia, Sweden. HPLC (Agilent Technologies, USA): P680 HPLC pump, ASI-100 autosampler, TCC-100 column oven, UVD170U UV detector (four wavelengths), quaternary eluent, online degasser, Chromeleon chromatography workstation. Preparative HPLC (Jiangsu Hanbang Technology Co., Ltd.): NP7005C pump, N3000D UV detector (dual wavelengths), semi-preparative dynamic mixer, EasyChrom-1000 chromatography workstation. Mass spectrometry was performed using an ultra-high performance liquid chromatography-tandem quadrupole electrostatic field orbital trap mass spectrometer (UHPLC-Q-Exactive Orbitrap MS, Thermo Fisher, MA, USA); nuclear magnetic resonance (NMR) was performed using a Bruker AVANCE NEO 600 NMR spectrometer (Bruker Corporation, USA); electronic balance (METTER AC-100, Sartorious, Germany); rotary evaporator (N-1001D, Shanghai Ailang Instrument Co., Ltd.); high-precision polarimeter (UAutopol VI, Rudolph, USA). Viola kunawurensis Royle Illustr, collected in June 2024 in Cele County, Hotan Prefecture, Xinjiang Uygur Autonomous Region, China, was identified by Associate Researcher Lu Chunfang of the Xinjiang Institute of Physics and Chemistry, Chinese Academy of Sciences as a plant of the genus Viola in the family Violaceae. The specimen is deposited in the National Basic Science Public Science Data Center - Arid Zone Medicinal Resources Database.
[0013] Example 1 a. After pulverizing 10.0 kg of raw Viola tianshanense whole herb, mix it thoroughly with a mixture of ammonia and acetone at room temperature to obtain the soaked medicinal material; b. At room temperature, the medicinal material obtained in step a is extracted by cold soaking in a mixed solution of chloroform and methanol, and the organic solvent is recovered by vacuum concentration to obtain an extract; c. Dissolve the extract obtained in step b in 5 times its volume of water, then extract it 5 times with the same volume of ethyl acetate as water. Take the ethyl acetate layer sample, concentrate it under reduced pressure to recover the ethyl acetate, and obtain the total alkaloids. d. The total alkaloids obtained in step c were separated by macroporous resin column chromatography. Gradient elution was performed using methanol-water with volume fractions of 10% to 70% as eluents. The eluted fractions were collected, and after thin-layer chromatography analysis, the same samples were combined to obtain 15 components, numbered sequentially as 1-15. Fraction 4 was separated by dextran gel column chromatography. Isocratic elution was performed using chloroform and methanol in a volume ratio of 1:2. The eluted samples were collected in fractions, and after thin-layer chromatography analysis, the same samples were combined and concentrated under reduced pressure to obtain the new compounds tianshan violacein C, tianshan violacein F and subfraction 4A. Fraction 5 was separated by silica gel column chromatography, eluted sequentially with dichloromethane and methanol in volume ratios of 19:1, 9:1, 17:3, and 8:2. The eluted samples were collected and concentrated to obtain three subfractions 5A-5C. 5A was purified by semi-preparative high-performance liquid chromatography with acetonitrile-water in a mobile phase of 20:70. Peaks with retention times of 30 min and 45 min were collected and concentrated to obtain compounds tianshan violacein A and tianshan violacein B. Fraction 6 was separated by polyamide column chromatography using 30% methanol-water as eluent for isocratic elution. The eluted samples were collected in fractions, and after thin-layer chromatography analysis, identical samples were combined to obtain four subfractions, numbered 6A-6B. Fraction 6A was purified by dextran gel column chromatography using methanol and acetone in a 1:1 volume ratio as eluent. The eluted samples were collected in fractions and concentrated to obtain compounds tianshan violacein D and tianshan violacein E.
[0014] Example 2 a. After pulverizing 10.0 kg of raw Viola tianshanense whole herb, mix it thoroughly with a mixture of ammonia and acetone at room temperature to obtain the soaked medicinal material; b. At room temperature, the medicinal material obtained in step a is extracted by cold soaking in a mixed solution of chloroform and methanol, and the organic solvent is recovered by vacuum concentration to obtain an extract; c. Dissolve the extract obtained in step b with 3 times the volume of water, then extract it 6 times with the same volume of ethyl acetate as water. Take the ethyl acetate layer sample, concentrate it under reduced pressure to recover the ethyl acetate, and obtain the total alkaloids. d. The total alkaloids obtained in step c were separated by polyamide method, and gradient elution was performed successively using ethanol and water with volume fractions of 10% to 70% as eluents. The eluted fractions were collected, and after thin-layer chromatography analysis, the same samples were combined to obtain 12 components, numbered 1-12 in sequence. Fraction 6 was separated by silica gel column chromatography, using petroleum ether and acetone in volume ratios of 19:1, 9:1, 17:3, and 8:2 as eluents for gradient elution. The eluted samples were collected, analyzed by thin-layer chromatography, and the same samples were combined to obtain compound corydaline A and 5 subfractions, numbered 6A-6E. Fraction 7 was separated by dextran gel column chromatography with chloroform and methanol in a volume ratio of 1:1 as the eluent. The lighter-colored or colorless samples were collected in fractions and the same components were combined by thin-layer chromatography to obtain two crystals, namely the compounds tianshan violacein B and tianshan violacein D. Fraction 8 was separated by silica gel column chromatography, using petroleum ether and acetone in a volume ratio of 19:1 to 8:2 as eluents for gradient elution. The eluted samples were collected and analyzed by thin-layer chromatography. The same samples were combined to obtain two subfractions, numbered 8A-8B. Fraction 8B was purified by semi-preparative high-performance liquid chromatography (HPLC) with acetonitrile-water in a volume ratio of 45:55. The peak with a retention time of 15 min was collected and concentrated to obtain the compound colophonine C. Fraction 9 was separated by macroporous resin column chromatography and eluted isocratically with a 15% (v / v) methanol aqueous solution. The eluted sample was collected and concentrated. The sample after removing pigment impurities was purified by semi-preparative high performance liquid chromatography with acetonitrile-water at a v / v ratio of 35:65. Peaks with retention times of 15 min and 35 min were collected and concentrated to obtain compounds tianshan violacein E and tianshan violacein F.
[0015] Example 3 a. After pulverizing 10.0 kg of raw Viola tianshanense whole herb, mix it thoroughly with a mixture of ammonia and acetone at room temperature to obtain the soaked medicinal material; b. At room temperature, the medicinal material obtained in step a is extracted by cold soaking in a mixed solution of chloroform and methanol, and the organic solvent is recovered by vacuum concentration to obtain an extract; c. Dissolve the extract obtained in step b in 4 times its volume of water, then extract it 6 times with the same volume of ethyl acetate as water. Take the ethyl acetate layer sample, concentrate it under reduced pressure to recover the ethyl acetate, and obtain the total alkaloids. c. The total alkaloid extract obtained in step c was separated by silica gel column chromatography. The eluents were successively used as petroleum ether and acetone in a volume ratio of 100:1 to 8:2. The eluted fractions were collected and analyzed by thin-layer chromatography. The same samples were combined to obtain 8 components, which were numbered 1-8 in sequence. Fraction 2 was separated by silica gel column chromatography, and gradient elution was performed using chloroform and acetone in volume ratios of 19:1 to 8:2. The eluted samples were collected, and after thin-layer chromatography analysis, the same samples were combined to obtain tianshan violacein F and 4 subfractions, numbered 2A-2D. Fraction 2A was separated by polyamide column chromatography and eluted isocratically with a 20% (v / v) methanol aqueous solution. The eluted sample was collected and concentrated, and then pigment impurities were removed by dextran gel column chromatography with methanol as the eluent. The lighter-colored or colorless samples were collected in fractions and concentrated to obtain compounds tianshan violacein A and tianshan violacein B. Fraction 4 was separated by polyamide column chromatography using a gradient elution with petroleum ether and ethyl acetate in volume ratios of 19:1, 9:1, 17:3, and 8:2. The eluted samples were collected and analyzed by thin-layer chromatography. Samples of the same type were combined to obtain three subfractions, numbered 4A-4C. Fraction 4B was purified by semi-preparative high-performance liquid chromatography (HPLC) using a mobile phase of methanol-water in a volume ratio of 20:80. The peak with a retention time of 32 min was collected and concentrated to obtain the compound colophonine D. Fraction 5 was separated by macroporous resin column chromatography, eluted with a gradient of 30%-60% (v / v) ethanol-water solution. The eluted sample was collected and concentrated, and the combined subfractions were detected by analytical high performance liquid chromatography and numbered 5A-5F. Pigment impurities in fraction 5A were removed by dextran gel column chromatography, with chloroform and acetone in a 1:2 (v / v) eluent. Lighter or colorless samples were collected and concentrated to obtain compounds Viola tianshanensis C-alkaloid and Viola tianshanensis E-alkaloid.
[0016] Example 4 a. After pulverizing 10.0 kg of raw Viola tianshanense whole herb, mix it thoroughly with a mixture of ammonia and acetone at room temperature to obtain the soaked medicinal material; b. At room temperature, the medicinal material obtained in step a is extracted by cold soaking in a mixed solution of chloroform and methanol, and the organic solvent is recovered by vacuum concentration to obtain an extract; c. Dissolve the extract obtained in step b in 5 times its volume of water, then extract it several times with the same volume of ethyl acetate as water. Take the ethyl acetate layer sample, concentrate it under reduced pressure to recover the ethyl acetate, and obtain the total alkaloids. d. The total alkaloids obtained in step c were separated by silica gel column chromatography. Gradient elution was performed using hexane and diethylamine in volume ratios of 100:1 to 8:2. The eluted fractions were collected and analyzed by thin-layer chromatography. The same samples were combined to obtain 10 components, which were numbered 1-10. Fraction 2 was separated by polyamide column chromatography, and chloroform and acetone in volume ratios of 19:1 to 8:2 were used as eluents for gradient elution. The eluted samples were collected, and after thin-layer chromatography analysis, the same samples were combined to obtain colophonine A. Fraction 3 was separated by macroporous resin column chromatography, eluted isocratically with 20% ethanol aqueous solution, the eluted sample was collected and concentrated, and then separated by dextran gel column chromatography with methanol as the eluent. The lighter or colorless samples were collected in fractions and concentrated to obtain compounds tianshan violacein B and tianshan violacein D. Fraction 4 was separated by polyamide column chromatography, using petroleum ether and ethyl acetate in a volume ratio of 19:1 to 8:2 as eluents for gradient elution. The eluted samples were collected, and after thin-layer chromatography analysis, identical samples were combined to obtain three subfractions, numbered 4A-4C. Fraction 4C was purified by semi-preparative high-performance liquid chromatography (HPLC) with a mobile phase of methanol-water in a volume ratio of 60:40. The peak with a retention time of 32 min was collected and concentrated to obtain the compound colophonine C. Fraction 5 was separated by macroporous resin column chromatography, eluted with a gradient of 10%-40% (v / v) ethanol-water solution. The eluted samples were collected and concentrated, and the combined subfractions were detected by analytical high performance liquid chromatography and numbered 5A-5C. Pigment impurities in fraction 5B were removed by polyamide column chromatography, with 20% (v / v) methanol-water as the eluent. The lighter-colored or colorless samples were collected and concentrated to obtain compounds tianshan violacein E and tianshan violacein F.
[0017] Example 5 a. After pulverizing 10.0 kg of raw Viola tianshanense whole herb, mix it thoroughly with a mixture of ammonia and acetone at room temperature to obtain the soaked medicinal material; b. At room temperature, the medicinal material obtained in step a is extracted by cold soaking in a mixed solution of chloroform and methanol, and the organic solvent is recovered by vacuum concentration to obtain an extract; c. Dissolve the extract obtained in step b in 5 times its volume of water, then extract it several times with the same volume of ethyl acetate as water. Take the ethyl acetate layer sample, concentrate it under reduced pressure to recover the ethyl acetate, and obtain the total alkaloids. d. The total alkaloids obtained in step c were separated by silica gel column chromatography. The elution was carried out by gradient elution with cyclohexane and acetone in volume ratios of 100:1 to 8:2. The eluted fractions were collected and analyzed by thin-layer chromatography. The same samples were combined to obtain 8 components, which were numbered 1-8 in sequence. Fraction 4 was separated by silica gel column chromatography, eluted sequentially with dichloromethane and acetone in volume ratios of 19:1, 9:1, 17:3, and 8:2. The eluted samples were collected and concentrated, and then pigment impurities were removed by polyamide column chromatography with 20% methanol-water as the eluent. Samples with lighter or colorless colors were collected. The samples after removing pigment impurities were purified by semi-preparative high-performance liquid chromatography with acetonitrile-water in a volume ratio of 25:75. Peaks with retention times of 25 min and 33 min were collected, respectively, and concentrated to obtain compounds tianshan violacein A and tianshan violacein B. Fraction 5 was separated by macroporous resin column chromatography using 30% methanol-water as eluent for isocratic elution. The eluted samples were collected in fractions, and after thin-layer chromatography analysis, identical samples were combined to obtain four subfractions, numbered 5A-5D. Fraction 5C was purified by dextran gel column chromatography using chloroform and acetone in a 1:1 volume ratio as eluent. The eluted samples were collected in fractions and concentrated to obtain the compound colophonine C. Fraction 6 was separated by dextran gel chromatography with methanol and acetone in a volume ratio of 2:1 as the eluent. The eluted sample was collected and concentrated, and then combined by thin-layer chromatography to obtain two subfractions, numbered 6A and 6B. 6A and 6B were purified by semi-preparative high-performance liquid chromatography with methanol-water in a volume ratio of 20:80 for both fractions. The peaks with retention times of 20 min and 28 min were collected and concentrated to obtain compounds tianshan violacein D and tianshan violacein E. Fraction 7 was separated by macroporous resin column chromatography, using gradient elution with methanol and water at volume fractions of 45%, 60%, 80%, and 100% respectively. The eluted fractions were collected and analyzed by analytical high-performance liquid chromatography, and then combined into four subfractions, numbered 7A-7D. Fraction 7D was separated by dextran gel chromatography with chloroform and acetone in a 1:1 volume ratio as the eluent. The eluted samples were collected in fractions and concentrated to obtain the compound colophonine F.
[0018] Example 6 a. After pulverizing 10.0 kg of raw Viola tianshanense whole herb, mix it thoroughly with a mixture of ammonia and acetone at room temperature to obtain the soaked medicinal material; b. At room temperature, the medicinal material obtained in step a is extracted by cold soaking in a mixed solution of chloroform and methanol, and the organic solvent is recovered by vacuum concentration to obtain an extract; c. Dissolve the extract obtained in step b in 5 times its volume of water, then extract it several times with the same volume of ethyl acetate as water. Take the ethyl acetate layer sample, concentrate it under reduced pressure to recover the ethyl acetate, and obtain the total alkaloids. d. The total alkaloids obtained in step c were separated by polyamide column chromatography. Gradient elution was performed using hexane and acetone in volume ratios of 100:1 to 8:2. The eluted fractions were collected, and after thin-layer chromatography analysis, the same samples were combined to obtain 9 components, numbered 1-9 in sequence. Fraction 3 was separated by silica gel column chromatography, eluted sequentially with chloroform and acetone gradients at volume ratios of 19:1, 9:1, and 17:3. The eluted samples were collected and concentrated, and then dextran gel column chromatography was used to remove pigment impurities. Methanol was used as the eluent, and samples with lighter or colorless colors were collected. The samples with removed pigment impurities were purified by semi-preparative high-performance liquid chromatography with acetonitrile-water at a volume ratio of 25:75. Peaks with retention times of 15 min and 19 min were collected, respectively, and concentrated to obtain compounds tianshan violacein B and tianshan violacein C. Fraction 4 was separated by macroporous resin column chromatography using 30% ethanol-water as eluent for isocratic elution. The eluted samples were collected in fractions, and after thin-layer chromatography analysis, identical samples were combined to obtain three subfractions, numbered 4A-4C. Fraction 4B was purified by dextran gel column chromatography using methanol and acetone in a 1:1 volume ratio as eluent. The eluted samples were collected in fractions and concentrated to obtain compounds tianshan violacein A and tianshan violacein D. Fraction 9 was separated by dextran gel chromatography with methanol, chloroform and acetone in a volume ratio of 1:1:1 as eluents. The eluted sample was collected and concentrated, and then combined by thin-layer chromatography to obtain two subfractions, numbered 9A and 9B. 9A and 9B were repeatedly purified by recrystallization to obtain compounds tianshan violacein E and tianshan violacein F.
[0019] Example 7 The use of the alkaloids isolated from Viola tianshanensis described in this invention in the preparation of analgesic drugs, using ICR mice as an example: Screening of analgesic activity of the obtained alkaloid compounds: Laboratory animals: ICR mice, weighing 20-30g, half male and half female, were provided by the Department of Experimental Animals, Xinjiang Medical University. The mice were housed in an SPF-grade animal experimental center with an indoor temperature of 24±2℃ and a relative humidity of 40%-70%. They had free access to water and food, but were fasted for 10 hours before administration of the drug, although water was allowed. Experimental instruments and reagents: Dimethyl sulfoxide (Adamas), glacial acetic acid (Aladdin), physiological saline; Experiment content: Drug solution preparation: Take 2.0 mg of sample, dissolve it in 5% DMSO, then dilute with physiological saline to prepare a concentration of 0.5 mg / ml. The model group serves as a physiological saline control and should be stored at room temperature, protected from light. Preparation of 0.6% acetic acid solution: 30 minutes before model preparation, prepare a 0.6% acetic acid solution and store it in an ice water box. 15 minutes before administration, place the 0.6% acetic acid solution in a 37°C water bath to maintain a constant temperature. Acetic acid writhing test in mice: Eighty-four ICR mice, half male and half female, were randomly divided into seven groups: a model group and each test drug group, with 12 mice in each group. All mice in each group received an intraperitoneal injection of 0.1 mL / 10 g of the drug, at a dose of 5 mg / kg. The model group received an equal volume of physiological saline. Thirty minutes after drug administration, all mice in each group received an intraperitoneal injection of 0.1 mL / 10 g of 0.6% glacial acetic acid. The number of writhing episodes within 30 minutes was recorded. The pain inhibition percentage of each drug group was compared with that of the model group. Pain inhibition percentage (%) = (number of writhing episodes in the model group - number of writhing episodes in the drug group) / number of writhing episodes in the model group × 100%. The experimental results are shown in Table 3: Table 3. Effects of alkaloid compounds in Viola tianshanense on acetic acid-induced pain in mice. Compared with the model group, .
[0020] To investigate the analgesic effect of violacein AF, an alkaloid compound isolated from Viola tianshanense, this study established a pain model in mice by intraperitoneal injection of acetic acid. The analgesic efficacy was comprehensively evaluated by simulating pain. The results showed that all six alkaloid compounds obtained from Viola tianshanense significantly reduced the number of writhing movements in the painful mice, with pain inhibition rates all exceeding 60%, demonstrating strong analgesic activity.
[0021] In summary, the results of this study provide a solid experimental basis for further exploring the potential of these compounds as clinical analgesics.
[0022] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.
Claims
1. An alkaloid compound, comprising the following compounds, the structural formulas of which are as follows: in: The name of Viola tianshanensis alkaloid A is: (S,Z)-4-(4-hydroxy-4,6,6-trimethyltetrahydropyrimidine-2(1H)-ylidene)-2,2,6-trimethylpyridine-3,5(2H,4H)-dione; The name of Tianshan Violane B is: 3,5,5,8,10,10-hexamethylpyrido[3,4-g]pyrrolo[2,1-a]isoquinoline-6,11(5H,10H)-dione; The name of Viola tianshanensis alkaloid C is: (Z)-2,2,4,6,6-pentamethyl-3-(2-oxopropylidene)-2,3,6,7-tetrahydro-5H-pyrrolo[2,3-b]pyridin-5-one; The name of tianshan violane D is: (1aR,2R,7R,8aS)-1a,3,3,6,6-pentamethyl-8-methylene-1a,2,4,6,7,8-hexahydro-3H-epoxy[2,3-e]quinoline-2,7-diol; The name of Viola tianshanensis alkaloid E is: (7S,8S)-7,8-dihydroxy-2,5,5,7-tetramethyl-7,8-dihydroindoleazine-6(5H)-one; The name of tianshan violane F is: (R)-7-hydroxy-2,5,5,7-tetramethyl-6,7-dihydroindoleazine-3(5H)-one.
2. The method for preparing an alkaloid compound according to claim 1, characterized in that, Includes the following steps: a. After pulverizing the whole herb of Viola tianshanensis, mix it thoroughly with a mixture of ammonia and acetone at room temperature to obtain the soaked medicinal material; b. At room temperature, the medicinal material obtained in step a is extracted by cold soaking in a mixed solution of chloroform and methanol, and the organic solvent is recovered by vacuum concentration to obtain an extract; c. Dissolve the extract obtained in step b in 3-5 times its volume of water, then extract it five times with the same volume of ethyl acetate as water. Take the ethyl acetate layer sample, concentrate it under reduced pressure to recover the ethyl acetate, and obtain the total alkaloids. d. The total alkaloids obtained in step c are separated by two, three, or four of the following methods: silica gel column chromatography, thin-layer chromatography, polyamide column chromatography, macroporous resin column chromatography, dextran gel LH-20 column chromatography, and high-performance liquid chromatography.
3. The method for preparing an alkaloid compound according to claim 2, characterized in that, The two separation methods in step d are as follows: The total alkaloid extract obtained in step c was separated by silica gel column chromatography. Gradient elution was performed using petroleum ether and diethylamine in volume ratios of 100:1, 50:1, 25:1, 19:1, 9:1, 17:3, and 8:
2. The eluted fractions were collected, and after thin-layer chromatography analysis, the same samples were combined to obtain 8 components, numbered 1-8 in sequence. Fraction 6 was further separated by silica gel column chromatography, using petroleum ether and acetone in volume ratios of 19:1, 9:1, 17:3, and 8:2 as eluents for gradient elution. The eluted samples were collected, and after thin-layer chromatography analysis, the same samples were combined to obtain 7 subfractions, numbered 6A-6G. Fraction 6B was used to remove pigment impurities by dextran gel column chromatography with chloroform and methanol in a 1:1 volume ratio as eluent. Samples with lighter or colorless color were collected. The samples with removed pigment impurities were purified by semi-preparative high performance liquid chromatography with acetonitrile-water in a 30:70 volume ratio as mobile phase. Peaks with retention times of 18 min and 25 min were collected and concentrated to obtain compounds tianshan violacein A and tianshan violacein B. Fraction 6E was separated by silica gel column chromatography using gradient elution with petroleum ether and ethyl acetate in volume ratios of 19:1, 9:1, 17:3, and 8:
2. The eluted samples were collected and analyzed by thin-layer chromatography. Samples of the same type were combined to obtain four subfractions, numbered 6Ea-6Ed. Fraction 6Ea was purified by semi-preparative high-performance liquid chromatography (HPLC) using acetonitrile-water in a mobile phase of 23:
77. The peak with a retention time of 21 min was collected and concentrated to obtain the compound colophonine C. The fraction 6F was separated by polyamide column chromatography and eluted isocratically with 30% methanol aqueous solution. The eluted sample was collected and concentrated. The sample was purified by semi-preparative high performance liquid chromatography to remove pigment impurities. The mobile phase was acetonitrile-water with a volume ratio of 15:
85. The peaks with retention times of 15 min and 22 min were collected and concentrated to obtain compounds tianshan violacein D and tianshan violacein E. Fraction 6G was used to remove pigment impurities by dextran gel column chromatography with methanol as the eluent. Samples with lighter or colorless color were collected. Fraction 6G after removing pigment impurities was then purified by semi-preparative high performance liquid chromatography with acetonitrile-water at a volume ratio of 18:82 as the mobile phase. The peak with a retention time of 31 min was collected and concentrated to obtain the compound colophonine F.
4. The method for preparing an alkaloid compound according to claim 2, characterized in that, The three separation methods in step d are as follows: The total alkaloid extract obtained in step c was separated by silica gel column chromatography. Gradient elution was performed using chloroform and methanol in volume ratios of 100:1, 50:1, 25:1, 19:1, 9:1, 17:3, and 8:
2. The eluted fractions were collected, and after thin-layer chromatography analysis, the same samples were combined to obtain 6 components, numbered 1-6 in sequence. Fraction 4 was further separated by silica gel column chromatography, using chloroform and methanol in volume ratios of 19:1, 9:1, 17:3, and 8:2 as eluents for gradient elution. The eluted samples were collected, and after thin-layer chromatography analysis, the same samples were combined to obtain four subfractions, numbered 4A-4D. Fraction 4A was separated by polyamide column chromatography and eluted isocratically with a 20% (v / v) methanol aqueous solution. The eluted sample was collected and concentrated, and then dextran gel column chromatography was used to remove pigment impurities. Methanol was used as the eluent. Samples with lighter color or no color were collected. The sample with removed pigment impurities was purified by semi-preparative high performance liquid chromatography with acetonitrile-water at a v / v ratio of 25:
75. Peaks with retention times of 25 min and 33 min were collected, respectively. After concentration, compounds tianshan violacein A and tianshan violacein B were obtained. Fraction 4B was separated by polyamide column chromatography using a gradient elution with petroleum ether and acetone in volume ratios of 19:1, 9:1, 17:3, and 8:
2. The eluted samples were collected and analyzed by thin-layer chromatography. Samples of the same type were then combined to obtain three subfractions, numbered 4Ba-4Bc. Fraction 4Ba was purified by semi-preparative high-performance liquid chromatography (HPLC) using acetonitrile-water in a mobile phase of 20:
80. The peak with a retention time of 25 min was collected and concentrated to obtain the compound colophonine C. Fraction 4C was separated by macroporous resin column chromatography, eluted with a gradient of 30%-80% methanol-water solution. The eluted sample was collected and concentrated, and the fractions were combined by analytical high-performance liquid chromatography to obtain four subfractions, numbered 4Ca-4Cd. Pigment impurities in fraction 4Cb were removed by dextran gel column chromatography, with methanol and acetone in a 1:1 volume ratio as the eluent. Samples with lighter or colorless colors were collected. The sample after removing pigment impurities was purified by semi-preparative high-performance liquid chromatography, with acetonitrile-water in a 18:82 volume ratio as the mobile phase. Peaks with retention times of 18 min and 26 min were collected, and the concentrations yielded compounds tianshan violacein D and tianshan violacein E. The 4D fraction was purified by dextran gel column chromatography to remove pigment impurities using methanol as the eluent. Lighter or colorless samples were collected and then purified by macroporous resin column chromatography using 15% ethanol-water as the eluent. The eluted samples were collected in fractions and concentrated to obtain the compound colophonine F and subfraction 4Da.
5. The method for preparing an alkaloid compound according to claim 2, characterized in that, The four separation methods in step d are as follows: The total alkaloid extract obtained in step c was separated by macroporous resin column chromatography. Gradient elution was performed using ethanol and water with volume fractions of 10%, 30%, 50%, 80%, 90%, and 100% as eluents. The eluted fractions were collected, and after thin-layer chromatography analysis, the same samples were combined to obtain 5 components, numbered 1-5 in sequence. Fraction 3 was separated by polyamide column chromatography, and gradient elution was performed using chloroform and methanol in volume ratios of 19:1, 9:1, 17:3, and 8:
2. The eluted samples were collected, and after thin-layer chromatography analysis, the same samples were combined to obtain four subfractions, numbered 3A-3D. Fraction 3A was separated by silica gel column chromatography, eluted sequentially with dichloromethane and acetone in volume ratios of 19:1, 9:1, 17:3, and 8:
2. The eluted samples were collected and concentrated, and then pigment impurities were removed by polyamide column chromatography with 20% methanol-water as the eluent. Samples with lighter or colorless colors were collected. The samples after removing pigment impurities were purified by semi-preparative high-performance liquid chromatography with acetonitrile-water in a volume ratio of 25:
75. Peaks with retention times of 25 min and 33 min were collected, respectively. After concentration, compounds tianshan violacein A and tianshan violacein B were obtained. Fraction 3B was separated by macroporous resin column chromatography using 30% methanol-water as eluent for isocratic elution. The eluted samples were collected in fractions, and after thin-layer chromatography analysis, identical samples were combined to obtain four subfractions, numbered 3Ba-3Bd. Fraction 3Bd was purified by dextran gel column chromatography using chloroform and acetone in a 1:1 volume ratio as eluent. The eluted samples were collected in fractions, concentrated, and yielded the compound corydaline C and subfractions 3Bd1 and 3Bd2. Fraction 3C was separated by dextran gel chromatography with methanol and acetone in a volume ratio of 2:1 as the eluent. The eluted sample was collected and concentrated, and then combined by thin-layer chromatography to obtain two subfractions, numbered 3Ca and 3Cb, respectively. 3Ca and 3Cb were purified by semi-preparative high-performance liquid chromatography with methanol-water in a volume ratio of 20:80 for both fractions. The peaks with retention times of 20 min and 28 min were collected, respectively, and then concentrated to obtain compounds tianshan violacein D and tianshan violacein E. Fraction 3D was separated by macroporous resin column chromatography using gradient elution with methanol and water at volume fractions of 45%, 60%, 80%, and 100%. The eluted fractions were collected and analyzed by analytical high-performance liquid chromatography, and then combined into four subfractions, numbered 3Da-3Dd. Fraction 3Dc was separated by dextran gel chromatography using chloroform and acetone at a volume ratio of 1:
1. The eluted samples were collected in fractions and concentrated to obtain the compound corydaline F and subfraction 3Dc1.
6. The use of the alkaloid compound according to claim 1 in the preparation of analgesic drugs.