CN115260208B - Lotus alkyl alkaloid compound and its preparation method and use - Google Patents

Lotus alkyl alkaloid compound and its preparation method and use Download PDF

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CN115260208B
CN115260208B CN202210927076.3A CN202210927076A CN115260208B CN 115260208 B CN115260208 B CN 115260208B CN 202210927076 A CN202210927076 A CN 202210927076A CN 115260208 B CN115260208 B CN 115260208B
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李宁
陈刚
周地
肖姣
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Shenyang Pharmaceutical University
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Abstract

Lotus alkane alkaloid compound and pharmaceutically acceptable salt, isomer or solvate thereof, and is characterized by having the following structural general formula (I) or (II):

Description

Lotus alkyl alkaloid compound and its preparation method and use
Technical Field
The invention relates to lotus seed alkane alkaloid compounds, a preparation method and application thereof, and belongs to the technical field of medicines.
Background
The faeces Vaccinium uliginosum (Stephania longa Lour) is a stephania japonica (Menispermaceae) herb vine, commonly known as Sapium sebiferum, trigonella foenum-graecum, etc. Is a common plant in the southern area of China, and is mainly distributed in the provinces of Yunnan, guangxi and the like, and grows in wet places such as roadsides, shrubs and the like. The whole plant of Du Jiu can be used as medicine, and has bitter taste and cold property. Has effects of dispelling pathogenic wind, activating collaterals, inducing diuresis, relieving swelling, clearing heat, and promoting diuresis.
In recent years, studies on chemical components and pharmacological actions of stephania plants have been increasing. Research shows that the plant is rich in alkaloid components, is an important material basis for exerting biological activity, such as stephanine (cepharanthine) for increasing white, tetrahydropalmatine (L-tetrahydropalmatine) for easing pain, tetrandrine (tetrandrine) with antibacterial and anti-inflammatory effects and the like are alkaloid components rich in stephania plant. Other scholars find that the plant also contains a small amount of flavone, anthraquinone and lignin components. However, the research on the chemical components and the pharmacological activity of the fecal sewage in the vaccinium uliginosum is not enough. In order to further enrich the structural diversity of the lotus alkyl alkaloid compounds, the structure-activity relationship of the components for resisting neuroinflammation is clarified. The chemical composition and pharmacological actions in the fecal skip and the like are systematically studied herein.
Disclosure of Invention
The invention aims at lotus alkyl alkaloid and pharmaceutically acceptable salt, isomer or solvate thereof, and particularly provides 11 novel alkaloid structures separated and identified from fecal skip and the preparation method and application thereof in developing medicaments for treating neurodegenerative diseases.
Lotus alkane alkaloid compound and pharmaceutically acceptable salt, isomer or solvate thereof, which has the following structural general formula (I) or (II):
R 1 is cinnamoyl, benzoyl, 4-methoxybenzoyl, C1-C4 acyl;
R 2 is hydroxy, C1-C4 alkoxy;
R 3 hydrogen, oxygen, cyano;
R 4 is hydrogen, C1-C4 alkyl;
R 5 is hydrogen or oxygen.
The alkaloid and pharmaceutically acceptable salts, isomers or solvates thereof have one of the following chemical structural formulas:
(6S,7S,8R,10S,13S,14S,17S)-stephalonine T(1)
(6S,7S,8R,10S,13S,14S)-stephalonine U(2)
(6S,7S,8R,10S,13S,14S,16R)-stephalonine V(3)
(6S,7S,8R,10S,13S,14S)-4-methoxyl-stephalonine F(4)
(6S,7S,8R,10S,13R,14S)-16-oxo-stephalonine F(5)
(6S,7S,8R,10S,13S,14S)-4,4'-dimethoxyl-stephalonine F(6)
(6S,7S,8R,10S,13R,14S)-4-methoxyl-16-oxo-stephalonine F(7)
(6S,7S,8R,10S,13S,14S)-6-O-acetyl-N-methylstephuline(8)
(2'R,6S,7S,8R,10S,13R,14S)-stephalonine W(9)
(2'R,6S,7S,8R,10S,13S,14S,17S)-stephalonine X(10)
(2'R,6S,7S,8R,10S,13S,14S)-stephalonine Y(11)。
another object of the present invention is to provide a method for preparing the lotus seed alkyl alkaloid compound. The method comprises the following steps:
extracting whole plants of the fecal Vaccinium uliginosum (Stephania longa) by ethanol aqueous solution, and recovering the extracting solution to obtain ethanol extract; dissolving the ethanol extract with water, adjusting pH to 2, adding petroleum ether to obtain extract A, adjusting pH of water layer to 8, and adding dichloromethane to obtain extract B; separating the obtained extract B by silica gel column chromatography, and gradient eluting with mixed solvent I; separating the obtained fraction by ODS chromatography, and gradient eluting with mixed solvent II as mobile phase; the obtained eluent is further separated by preparative HPLC-UV, and the compound 1-11 is obtained by gradient elution with mixed solvent of methanol and water 4:6-9:1 or acetonitrile and water 2:8-8:2 as mobile phase,
the mixed solvent I is a mixed solvent of petroleum ether and ethyl acetate, a mixed solvent of petroleum ether and acetone, a mixed solvent of chloroform and acetone, a mixed solvent of dichloromethane and acetone, a mixed solvent of chloroform and methanol or a mixed solvent of dichloromethane and methanol; the mixed solvent II is methanol and water 1:9 to 9:1 mixed solvent, acetonitrile and water 1: 9-8: 2 mixing the solvents.
In the method, the whole plant of the fecal skip is extracted for 3 to 6 times by heating reflux or cold leaching with ethanol water to obtain the ethanol extract, wherein the volume concentration of the ethanol is 60 to 100 percent, and the fecal skip is: the weight-volume ratio of the ethanol is 1:8-1:15 g/mL.
In the method, after the extract of the fecal skip is dissolved in water, the acid-soluble alkali-precipitation method is adopted for treatment, the pH is regulated to 2 by 4.5 percent HCl, and petroleum ether is added to obtain an extract A; further adding 5% Na to the aqueous layer 2 CO 3 The pH was adjusted to 8 and methylene chloride was added to give extract B. Each solvent is extracted for 3 to 6 times, and the volume ratio of the water phase to the organic phase is 1:1.
In the method, the mixed solvent I is a mixed solvent consisting of petroleum ether and ethyl acetate according to the volume ratio of 100:10-1:1, or is a mixed solvent consisting of petroleum ether and acetone according to the volume ratio of 100:10-1:1, or is methylene dichloride and acetone according to the volume ratio of 100: 3-100: 8, or chloroform and acetone according to the volume ratio of 100: 3-100: 8, or dichloromethane and methanol in a volume ratio of 100:0 to 100:8, or the volume ratio of chloroform to methanol is 100:0 to 100: 8.
In the method, the mixed solvent II is methanol and water in a ratio of 3:7-8: 2, mixing the solvent, acetonitrile and water in a ratio of 1:9-6: 4, mixing the solvents.
In the above method, the mobile phase of the preparative HPLC-UV chromatography is methanol and water 4: 6-8: 2, mixing the solvents; or acetonitrile and water 3: 7-7: 3 mixing the solvents.
It is still another object of the present invention to provide a pharmaceutical composition comprising the lotus-alkane-type alkaloid compound and pharmaceutically acceptable salts, isomers or solvates thereof.
A pharmaceutical composition comprises the lotus seed alkane alkaloid compound, pharmaceutically acceptable salts, isomers or solvates thereof and a pharmaceutically acceptable carrier.
Meanwhile, the invention provides application of the lotus seed alkyl alkaloid and pharmaceutically acceptable salts, isomers or solvates thereof or a pharmaceutical composition thereof in preparing medicaments for preventing or treating neurodegenerative diseases.
The beneficial effects of the invention are as follows: the invention provides lotus seed alkyl alkaloid compounds prepared by taking fecal skip and vaccinium uliginosum as raw materials, and specifically prepares and identifies 11 lotus seed alkyl alkaloid compounds. Meanwhile, the invention evaluates the anti-neuroinflammation activity of the prepared lotus alkyl alkaloid by using an LPS-induced microglial cell overactivation model. The results show that 3,8, 10 can obviously inhibit BV2 microglial cells induced by LPS from releasing NO under the condition of not affecting the survival rate of microglial cells BV2, and shows a certain anti-neuroinflammation effect. Therefore, the lotus-alkane-type alkaloid prepared in the invention can be applied to the development of medicaments for treating neurodegenerative diseases.
Detailed Description
The following non-limiting examples will enable those of ordinary skill in the art to more fully understand the invention and are not intended to limit the invention in any way.
The test methods described in the following examples, unless otherwise specified, are all conventional; the reagents and materials, unless otherwise specified, are commercially available.
Example 1
(1) Reflux-extracting 20kg of whole plant of Vaccinium uliginosum with 80% ethanol under heating for 3 times and 2 hr each time (200L each time), and recovering the extractive solution under reduced pressure to obtain ethanol extract;
(2) Dissolving the ethanol extract obtained in the step (1) by water to obtain an extract, adjusting the pH to 2 by using 4.5% HCl, and adding petroleum ether to obtain an extract A. Further adding 5% Na to the aqueous layer 2 CO 3 Adjusting pH to 8, and adding dichloromethane to obtain extract B; the volume ratio of the extracting solution to the extracting agent is 1:1, and each extracting agent is extracted for 3 times;
(3) Separating the dichloromethane extract B obtained in the step (2) by silica gel column chromatography, eluting with a mixed solvent of petroleum ether and ethyl acetate of 100:10, 100:15,100:20,100:40 and 100:100;
(4) Separating the 100:10-100:20 flow obtained in the step (3) by ODS chromatography, and performing gradient elution by using 10:90,30:70, 50:50, 70:30, 80:20 and 90:10 of methanol-water as mobile phases;
(5) The methanol-water (50:50-80:20) fraction obtained in the step (4) was prepared by HPLC-UV chromatography, the flow rate was 4mL/min, the mobile phase was methanol: water=60:40, compound 2 (t R =40 min) (yield 0.0001%), 3 (t R =23 min) (yield 0.00003%), 4 (t R =27 min) (yield 0.000015%), 6 (t R =29 min) (yield 0.000009%), 8 (t) R =19 min) (yield 0.00002%), 9 (t R =43 min) (yield 0.000001%);
(6) Separating the methanol-water (70:30-90:10) fraction obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm, and taking a mixed solvent of 75:25 methanol-water as a mobile phase at a flow rate of 4mL/min to obtain a compound 1 (t) R =33 min) (yield 0.00001%), 5 (t R =37 min) (yield 0.00006%), 7 (t) R =39 min) (yield 0.000018%), 10 (t R =41 min) (yield 0.000006%), 11 (t R =47 min) (yield 0.000008%).
The structure of the new lotus alkane type alkaloid 1-11 is identified according to the physicochemical properties and spectrum data of the new lotus alkane type alkaloid.
The structural identification data of lotus alkyl alkaloid 1 are as follows:
white powder (CH) 3 OH), improving the positive reaction of bismuth potassium iodide,HR-ESI-MS gives an excimer ion peak [ M+H ]] + m/z：538.2447(calcd.538.2441 for C 30 H 36 NO 8 ) To which it is bonded 1 H-NMR (see Table 1) and 13 the C-NMR data (see Table 3) speculate that the molecular formula is C 30 H 35 NO 8 The unsaturation was 14. 1 The coupling constants of H-6 and H-7 on the H-NMR spectrum were 4.2Hz, and thus they were determined to be in the cis configuration. H-7 was observed to correlate with H-5a in the NOESY spectrum; h-5a is associated with H-15 a; h-15b, H-16a being related to H-9 b; h-9a and 17-CH 3 Relatedly, hydrogen at positions 6 and 7 and positions 13 and 14 are indicatedThe ethylamine bridges are spatially oriented identically, and the relative configuration of compound 1 is 6S,7S,8R,10S,13S,14S, 17S. The measured ECD of compound 1 fitted well to the 6S,7S,8R,10S,13S,14S configuration, suggesting that compound 1 is (6S, 7S,8R,10S,13S,14S, 17S) -stephlonine T.
The structural identification data of lotus alkyl alkaloid 2 are as follows:
white amorphous powder (CH) 3 OH), improving the positive reaction of bismuth potassium iodide,HR-ESI-MS gives an excimer ion peak [ M ]] + m/z：522.2486(calcd.522.2492for C 30 H 36 NO 7 + ) To which it is bonded 1 H-NMR (see Table 1) and 13 the C-NMR data (see Table 3) speculate that the molecular formula is C 30 H 36 NO 7 + The unsaturation was 13. 1 The coupling constants of H-6 and H-7 on the H-NMR spectrum were 4.2Hz, and thus they were determined to be in the cis configuration. H-7 was observed to correlate with H-5b in the NOESY spectrum; h-5b is associated with H-16 a; h-16b, H-9b and 17-CH 3 In relation, the relative configuration of compound 2 was suggested to be 6S,7S,8R,10S,13S, 14S. The measured ECD of compound 2 fitted well to the 6s,7s,8r,10s,13s,14s configuration, suggesting that compound 2 is (6 s,7s,8r,10s,13s,14 s) -stephlonine U.
The structural identification data of lotus alkyl alkaloid 3 are as follows:
white powder (CH) 3 OH), improving the positive reaction of bismuth potassium iodide,HR-ESI-MS gives an excimer ion peak [ M+H ]] + m/z:547.2451(calcd.547.2444 for C 31 H 35 N 2 O 7 ) To which it is bonded 1 H-NMR (see Table 1) and 13 the C-NMR data (see Table 3) speculate that the molecular formula is C 31 H 34 N 2 O 7 The unsaturation was 16. 1 The coupling constants of H-6 and H-7 on the H-NMR spectrum were 4.5Hz, which was determined to be the cis configuration. H-7 was observed to correlate with H-5b in the NOESY spectrum; h-5b is associated with H-15 a; h-15b, H-16 andh-9a is related, suggesting that the hydrogens at positions 6,7 and the ethylamine bridges at positions 13, 14 are spatially oriented the same, and the relative configuration of compound 3 is 6S,7S,8R,10S,13S,14S, 16R. The measured ECD of compound 3 fitted well to the 6S,7S,8R,10S,13S,14S configuration, suggesting that compound 3 is (6S, 7S,8R,10S,13S,14S, 16R) -stephlonine V.
The structural identification data of lotus alkyl alkaloid 4 are as follows:
white powder (CH) 3 OH), improving the positive reaction of bismuth potassium iodide,HR-ESI-MS gives an excimer ion peak [ M+H ]] + m/z：496.2330(calcd.496.2335 for C 28 H 34 NO 7 ) To which it is bonded 1 H-NMR (Table 1) and 13 the C-NMR (Table 3) data speculates that the molecular formula is C 28 H 33 NO 7 The unsaturation was 13. 1 The coupling constants of H-6 and H-7 on the H-NMR spectrum were 3.9Hz, and thus they were determined to be in the cis configuration. H-7 was observed to correlate with H-5b in the NOESY spectrum; h-5b is associated with H-16 a; h-16b was associated with H-9a, suggesting that the hydrogens at positions 6,7 and the ethylamine bridges at positions 13, 14 are spatially oriented identically, and the relative configuration of compound 4 was 6S,7S,8R,10S,13S, 14S. The measured ECD of compound 4 fitted well to the 6S,7S,8R,10S,13S,14S configuration, suggesting that the absolute configuration of compound 4 is (6S, 7S,8R,10S,13S, 14S) -4-methoxyl-stephanine F.
The structural identification data of lotus alkyl alkaloid 5 are as follows:
pale yellow powder (CH) 3 OH), improving the positive reaction of bismuth potassium iodide,HR-ESI-MS gives an excimer ion peak [ M+H ]] + m/z：496.1967(calcd.496.1971 for C 27 H 30 NO 8 ) To which it is bonded 1 H-NMR (see Table 1) and 13 the C-NMR data (see Table 3) speculate that the molecular formula is C 27 H 29 NO 8 The unsaturation was 14. 1 The coupling constants of H-6 and H-7 on the H-NMR spectrum were 4.3Hz, and thus they were determined to be in the cis configuration. Observed in NOESY spectraH-7 and H-5b,17-NCH 3 Correlation; h-5b is associated with H-15 a; h-15b is associated with H-9b, suggesting that the hydrogens at positions 6,7 are spatially oriented the same as the ethylamine bridges at positions 13, 14, and the relative configuration of compound 5 is 6S,7S,8R,10S,13R, 14S. The measured ECD of compound 5 fitted well to the 6S,7S,8R,10S,13R,14S configuration, suggesting that compound 5 is (6S, 7S,8R,10S,13R, 14S) -16-oxo-stephanine F.
The structural identification data of lotus alkyl alkaloid 6 are as follows:
pale yellow powder (CH) 3 OH), improving the positive reaction of bismuth potassium iodide,HR-ESI-MS gives an excimer ion peak [ M+H ]] + m/z：526.2435(calcd.526.2441for C 29 H 36 NO 8 ) To which it is bonded 1 H-NMR (see Table 1) and 13 the C-NMR data (see Table 3) speculate that the molecular formula is C 29 H 35 NO 8 The unsaturation was 13. 1 The coupling constants of H-6 and H-7 on the H-NMR spectrum were 4.5Hz, and thus they were determined to be in the cis configuration. H-7 and H-5b,17-NC were observed in NOESY spectraH 3 Correlation; h-5b is associated with H-15 a; h-15b is associated with H-9b, suggesting that the hydrogens at positions 6,7 are spatially oriented the same as the ethylamine bridges at positions 13, 14, and the relative configuration of compound 6 is 6S,7S,8R,10S,13S, 14S. The measured ECD of compound 6 fits well with the 6S,7S,8R,10S,13S,14S configuration, indicating that the absolute configuration of compound 6 is 6S,7S,8R,10S,13S,14S, designated (6S, 7S,8R,10S,13S, 14S) -4,4' -dimethoxy-stephanine F.
The structural identification data of lotus alkyl alkaloid 7 are as follows:
pale yellow powder (CH) 3 OH), improving the positive reaction of bismuth potassium iodide,HR-ESI-MS gives an excimer ion peak [ M+Na ]] + m/z：532.1942(calcd.532.1947 for C 28 H 31 NO 8 Na), combined with it 1 H-NMR (see Table 1) and 13 the C-NMR data (see Table 3) speculate that the molecular formula is C 28 H 31 NO 8 The unsaturation was 14. 1 The coupling constants of H-6 and H-7 on the H-NMR spectrum were 4.4Hz, and thus they were determined to be in the cis configuration. H-7 and H-5b,17-NC were observed in NOESY spectraH 3 Correlation; h-5b is associated with H-15 a; h-15b is associated with H-9b, suggesting that the hydrogens at positions 6,7 are spatially oriented the same as the ethylamine bridges at positions 13, 14, the relative configuration of compound 7 is 6S,7S,8R,10S,13R, 14S. The measured ECD of compound 7 fitted well to the 6S,7S,8R,10S,13R,14S configuration, suggesting that compound 7 is (6S, 7S,8R,10S,13R, 14S) -4-methoxyl-16-oxo-stephanine F.
The structural identification data of lotus alkyl alkaloid 8 are as follows:
pale yellow powder (CH) 3 OH), improving the positive reaction of bismuth potassium iodide,HR-ESI-MS gives an excimer ion peak [ M+H ]] + m/z：420.2017(calcd.420.2022 for C 22 H 30 NO 7 ) To which it is bonded 1 H-NMR (see Table 2) and 13 the C-NMR data (see Table 3) speculate that the molecular formula is C 22 H 29 NO 7 The unsaturation was 9. 1 The coupling constants for critical hydrogens in the H-NMR spectrum and NOESY spectrum were determined. 1 The coupling constants of H-6 and H-7 on the H-NMR spectrum were 4.3Hz, and thus they were determined to be in the cis configuration. H-7 and H-5b,17-NC were observed in NOESY spectraH 3 Correlation; h-5b is associated with H-15 a; h-15b is related to H-9b, suggesting that the compound has the same spatial orientation of hydrogen at positions 6,7 and ethylamine bridges at positions 13, 14, with relative configurations of 6S,7S,8R,10S,13S, 14S. The measured ECD of compound 8 fitted well to the 6S,7S,8R,10S,13S,14S configuration, suggesting that compound 8 is (6S, 7S,8R,10S,13S, 14S) -6-O-acetyl-N-methyl-thiophiline.
The structural identification data of lotus alkyl alkaloid 9 are as follows:
pale yellow powder (CH) 3 OH), improving the positive reaction of bismuth potassium iodide,HR-ESI-MS gives an excimer ion peak [ M+Na ]] + m/z：498.2098(calcd.498.2104 for C 25 H 33 NO 8 Na), combined with it 1 H-NMR (see Table 2) and 13 the C-NMR data (see Table 3) speculate that the molecular formula is C 25 H 33 NO 8 The degree of unsaturation was 10. 1 The coupling constants of H-6 and H-7 on the H-NMR spectrum were 4.4Hz, and thus they were determined to be in the cis configuration. H-7 and H-5b,17-NC were observed in NOESY spectraH 3 Correlation; h-5b is associated with H-15 a; h-15b is associated with H-9b, suggesting that the hydrogens at positions 6,7 are spatially oriented the same as the ethylamine bridges at positions 13, 14, and the relative configuration of compound 9 is 6S,7S,8R,10S,13R, 14S. The configuration of the 2-methylbutyric acid fragment in the structure was further determined by an alkaline hydrolysis experiment, and the fragment in the structure was identified as a (2' r) -methylbutyric acid compound. The measured ECD of compound 9 fits well with the 2'R,6S,7S,8R,10R,13S,14S configuration, identifying compound 9 as (2' R,6S,7S,8R,10S,13R, 14S) -stephlonines W.
The structural identification data of lotus alkyl alkaloid 10 are as follows:
pale white amorphous powder (CH) 3 OH), improving the positive reaction of bismuth potassium iodide,HR-ESI-MS gives an excimer ion peak [ M+H ]] + m/z：492.2594(calcd.492.2597 for C 26 H 38 NO 8 ) Is combined with 1 H-NMR (see Table 2) and 13 the C-NMR data (see Table 3) speculate that the molecular formula is C 26 H 37 NO 8 The unsaturation was 9. 1 The coupling constants of H-6 and H-7 on the H-NMR spectrum were 4.2Hz, and thus they were determined to be in the cis configuration. H-7 was observed to correlate with H-5a in the NOESY spectrum; h-5a is associated with H-15 a; h-15b is associated with H-9 b; h-9a and 8-OCH 3 ，17-CH 3 Remote correlation suggests that the hydrogens at positions 6,7 and the ethylamine bridges at positions 13, 14 are spatially oriented identically, and the relative configuration of compound 10 is 6S,7S,8R,10S,13S, 14S. The configuration of the 2-methylbutyric acid fragment in the structure was further determined by an alkaline hydrolysis experiment, and the fragment in the structure was identified as a (2' r) -methylbutyric acid compound. The measured ECD of compound 10 was well fitted to the 2'R,6S,7S,8R,10S,13S,14S,17S configuration, identifying compound 10 as (2' R,6S,7S,8R,10S,13S, 14)S,17S)-stephalonines X。
The structural identification data of lotus alkyl alkaloid 11 are as follows: pale white amorphous powder (CH) 3 OH), improving the positive reaction of bismuth potassium iodide,HR-ESI-MS gives an excimer ion peak [ M ]] + m/z：476.2643(calcd.476.2643for C 26 H 38 NO 7 + ) To which it is bonded 1 H-NMR (see Table 2) and 13 the C-NMR data (see Table 3) speculate that the molecular formula is C 26 H 38 NO 7 + The unsaturation was 13. 1 The coupling constants of H-6 and H-7 on the H-NMR spectrum were 4.6Hz, and thus they were determined to be in the cis configuration. H-7 was observed to correlate with H-5b in the NOESY spectrum; h-5b is associated with H-16 a; h-16b was associated with H-9b, suggesting that the hydrogens at positions 6,7 and the ethylamine bridges at positions 13, 14 were spatially oriented identically, and the relative configuration of compound 11 was 6S,7S,8R,10S,13S, 14S. The configuration of the 2-methylbutyric acid fragment in the structure was further determined by an alkaline hydrolysis experiment, and the fragment in the structure was identified as a (2' r) -methylbutyric acid compound. The measured ECD of compound 11 fitted well to the 2'R,6S,7S,8R,10S,13S,14S configuration, identifying compound 11 as (2' R,6S,7S,8R,10S,13S, 14S) -stephlonines Y.
Example 2
(1) 20kg of the whole plant of the fecal dustpan and the Vaccinium uliginosum is subjected to cold soaking with 90% ethanol for 4 times, each time for 24 hours (each time using 300L), and the extracting solution is recovered under reduced pressure to obtain a crude extract;
(2) Dissolving the ethanol extract obtained in the step (1) by water, adjusting the pH to 2 by using 4.5% HCl, and adding petroleum ether to obtain an extract A. Further adding 5% Na to the aqueous layer 2 CO 3 Adjusting pH to 8, adding dichloromethane to obtain extract B, extracting with each solvent for 4 times at a volume ratio of extractive solution to extraction solvent of 1:1 to obtain different extracts;
TABLE 2 the 1 H NMR data for 8-11(J in Hz,600MHz)
TABLE 3 the 13 C NMR data for 1-11(150MHz)
(3) Separating the dichloromethane extract B obtained in the step (2) by silica gel column chromatography, eluting with petroleum ether and ethyl acetate mixed solvents of 00:100,100:15,100:20,100:40 and 100:100;
(4) Separating the 100:10-100:15 fraction obtained in the step (3) by ODS chromatography, and performing gradient elution by using 10:90 methanol/water, 30:70, 50:50, 70:30, 80:20 and 90:10 as mobile phases;
(5) The methanol-water (50:50-80:10) fraction obtained in the step (4) was prepared by HPLC-UV chromatography, the flow rate was 4mL/min, the mobile phase was methanol: water=60:40, compound 2 (t R =40 min) (yield 0.0001%), 3 (t R =23 min) (yield 0.00003%), 4 (t R =27 min) (yield 0.000015%), 6 (t R =29 min) (yield 0.000009%), 8 (t) R =19 min) (yield 0.00002%), 9 (t R =43 min) (yield 0.000001%);
(6) Separating the methanol-water (70:30-90:10) fraction obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm, and taking a mixed solvent of 75:25 methanol-water as a mobile phase at a flow rate of 4mL/min to obtain a compound 1 (t) R =33 min) (yield 0.00001%), 5 (t R =37 min) (yield 0.00006%), 7 (t) R =39 min) (yield 0.000018%), 10 (t R =41 min) (yield 0.000006%), 11 (t R =47 min) (yield 0.000008%).
The structural data for compounds 1-11 are identified in example 1.
Example 3
(1) 20kg of the whole plant of the fecal sewage is subjected to cold soaking for 4 times by using 100% ethanol, each time for 24 hours (each time using 160L), and the extracting solution is recovered under reduced pressure to obtain an ethanol extract;
(2) Dissolving the ethanol extract obtained in the step (1) by water to obtain an extract, adjusting the pH to 2 by using 4.5% HCl, and adding petroleum ether to obtain an extract A. Further adding 5% Na to the aqueous layer 2 CO 3 Adjusting pH to 8, and adding dichloromethane to obtain extract B; the volume ratio of the extracting solution to the extracting solvent is 1:1, and each solvent is extracted for 5 times to obtain extracts of different parts;
(3) Separating the dichloromethane extract B obtained in the step (2) by silica gel column chromatography, eluting with a mixed solvent of dichloromethane and methanol of 100:0, 100:1, 100:3, 100:5, 100:7 and 100:10;
(4) Separating the 100:1-100:7 flow obtained in the step (3) by ODS chromatography, and performing gradient elution by using acetonitrile/water 10:90,30:70, 60:40 and 80:20 as mobile phases;
(5) Separating acetonitrile/water (40:60-80:20) fraction obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm with a flow rate of 4mL/min, and eluting with acetonitrile/water (52:48) as mobile phase to obtain compound 2 (t) R =36 min) (yield 0.0001%), 3 (t R =17 min) (yield 0.00003%), 4 (t R =24 min) (yield 0.000015%), 6 (t R =25 min) (yield 0.000009%), 8 (t) R =15 min) (yield 0.00002%), 9 (t R =40 min) (yield 0.000001%);
(6) Separating acetonitrile/water (60:40-80:20) fraction obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm, and taking acetonitrile/water (61:39) as a mobile phase at a flow rate of 4mL/min to obtain a compound 1 (t) R =29 min) (yield 0.00001%), 5 (t R =32 min) (yield 0.00006%), 7 (t R =34 min) (yield 0.000018%), 10 (t R =39 min) (yield 0.000006%), 11 (t R =43 min) (yield 0.000008%).
The structural data for compounds 1-11 are identified in example 1.
Example 4
(1) Reflux-extracting 20kg of whole plant of faeces Vaccinium uliginosum with 70% ethanol under heating for 5 times and 2 hr each time (240L each time), and recovering the extractive solution under reduced pressure to obtain ethanol extract;
(2) Dissolving the ethanol extract obtained in the step (1) by water to obtain an extract, adjusting the pH to 2 by using 4.5% HCl, and adding petroleum ether to obtain an extract A. Further adding 5% Na to the aqueous layer 2 CO 3 Adjusting pH to 8, and adding dichloromethane to obtain extract B; the method comprises the steps of carrying out a first treatment on the surface of the The volume ratio of the extracting solution to the extracting solvent is 1:1, and each solvent is extracted for 6 times to obtain extracts of different parts;
(3) Separating the dichloromethane extract B obtained in the step (2) by silica gel column chromatography, eluting with a mixed solvent of chloroform and methanol of 100:0, 100:1, 100:3, 100:5, 100:7 and 100:10;
(4) Separating the 100:1-100:7 fraction obtained in the step (3) by ODS chromatography, and performing gradient elution by using 10:90,30:70, 50:50, 70:30, 80:20 and 90:10 of methanol and water as mobile phases;
the methanol-water (50:50-80:10) fraction obtained in the step (4) was prepared by HPLC-UV chromatography, the flow rate was 4mL/min, the mobile phase was methanol: water=60:40, compound 2 (t R =40 min) (yield 0.0001%), 3 (t R =23 min) (yield 0.00003%), 4 (t R =27 min) (yield 0.000015%), 6 (t R =29 min) (yield 0.000009%), 8 (t) R =19 min) (yield 0.00002%), 9 (t R =43 min) (yield 0.000001%);
(6) Separating the methanol-water (70:30-90:10) fraction obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm, and taking a mixed solvent of 75:25 methanol-water as a mobile phase at a flow rate of 4mL/min to obtain a compound 1 (t) R =33 min) (yield 0.00001%), 5 (t R =37 min) (yield 0.00006%), 7 (t) R =39 min) (yield 0.000018%), 10 (t R =41 min) (yield 0.000006%), 11 (t R =47 min) (yield 0.000008%).
The structural data for compounds 1-11 are identified in example 1.
Example 5
(1) Reflux-extracting 20kg of faeces Vaccinium uliginosum with 60% ethanol under heating for 6 times and 2 hr each time (200L each time), and recovering the extractive solution under reduced pressure to obtain ethanol extract;
(2) Step (1) obtaining BDissolving the alcohol extract with water to obtain extractive solution, adjusting pH to 2 with 4.5% HCl, and adding petroleum ether to obtain extract A. Further adding 5% Na to the aqueous layer 2 CO 3 Adjusting pH to 8, and adding dichloromethane to obtain extract B; the volume ratio of the extracting solution to the extracting solvent is 1:1, and each solvent is extracted for 5 times to obtain extracts of different parts;
(3) Separating the dichloromethane extract B obtained in the step (2) by silica gel column chromatography, eluting with a mixed solvent of dichloromethane and methanol of 100:0, 100:1, 100:3, 100:5, 100:7 and 100:10;
(4) Separating the 100:1-100:7 flow obtained in the step (3) by ODS chromatography, and performing gradient elution by using acetonitrile/water 10:90,30:70, 60:40 and 80:20 as mobile phases;
(5) Separating acetonitrile/water (40:60-80:20) fraction obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm with a flow rate of 4mL/min, and eluting with acetonitrile/water (52:48) as mobile phase to obtain compound 2 (t) R =36 min) (yield 0.0001%), 3 (t R =17 min) (yield 0.00003%), 4 (t R =24 min) (yield 0.000015%), 6 (t R =25 min) (yield 0.000009%), 8 (t) R =15 min) (yield 0.00002%), 9 (t R =40 min) (yield 0.000001%);
(6) Separating acetonitrile/water (60:40-90:10) fraction obtained in the step (4) by HPLC-UV chromatography, detecting at 210nm with flow rate of 4mL/min, and taking acetonitrile/water (61:39) as mobile phase to obtain compound 1 (t) R =29 min) (yield 0.00001%), 5 (t R =32 min) (yield 0.00006%), 7 (t R =34 min) (yield 0.000018%), 10 (t R =39 min) (yield 0.000006%), 11 (t R =43 min) (yield 0.000008%).
The structural data for compounds 1-11 are identified in example 1.
Microglial cell overactivation inhibition activity assay for compounds 1-11 prepared in examples 1-5:
(1) Experimental principle: the chronic inflammatory reaction mediated by microglial cell activation is an important link in the occurrence and development processes of neurodegenerative diseases, and inhibition of microglial cell activation can become a new target for drug discovery. LPS activates microglia to release NO, pro-inflammatory cytokines, reactive oxygen species, etc. The experiment evaluates the anti-inflammatory activity of the novel lotus alkyl alkaloid compounds 1-11 obtained from the fecal-friendly method by establishing a screening model for activating BV2 microglial cell abnormal activation by in vitro LPS and taking the activated microglial cell to release NO as an index.
(2) The experimental method comprises the following steps:
(1) culture of the mouse microglial cell line BV2
All glassware and metal instruments (culture flasks, pipettes, solution bottles, etc.) used in cell culture and model building were autoclaved at 121 ℃ for 30min to thoroughly remove contaminating LPS. Cell culture liquid containing 10% fetal bovine serum is prepared by taking DMDM culture medium as a base. Microglial cell at about 2×10 5 The concentration of cells/ml was 5% CO 2 Subculturing in culture flask at 37deg.C until the adherent cells occupy 70-80% of the bottom area of culture flask about the third day, digesting adherent cells with pancreatin, and subculturing to another culture flask. BV2 after being frozen and recovered by an ultralow temperature refrigerator at the temperature of minus 80 ℃ is used as the first generation, and BV2 cells of 3 rd generation to 8 th generation are selected for experiments.
(2) Medicine preparation method
All three compounds were in powder form and dissolved in DMSO. Stock was prepared at a concentration of 100mM and stored at-20 ℃. The culture medium is diluted by DMEM culture medium for clinical use, and the final concentration of DMSO is less than 1 mill, wherein the dilution is 100 mu M, 30 mu M, 10 mu M and 1 mu M in sequence.
(3) Griess assay for inhibition of LPS-activated microglia by compounds
Taking BV2 microglial cells in logarithmic growth phase, adjusting cell density to 0.2X10 by fresh DMEM medium containing 10% fetal bovine serum 5 cells/ml, inoculated in 96-well plates, 100. Mu.l/well, at 37℃in 5% CO 2 Is cultured in an incubator. After 24 hours of cell wall-attached culture, the cells are replaced by fresh culture solution without serum, and meanwhile, the dosing treatment is carried out. The 3 compounds were combined with LPS at doses 1, 10, 30, 100. Mu.M. And a blank control is set. The final LPS concentration in each of the dosing groups was 100ng/ml. After the cells are continuously cultured for 24 hours after the medicine is added, collecting supernatant, GDetection of NO in supernatant by rie colorimetric method 2- The content is as follows.
(4) MTT method for detecting influence of compound on microglial cell survival rate
Taking BV2 microglial cells cultured in logarithmic growth phase, and adjusting cell density to 2×10 with fresh DMEM medium containing 10% foetal calf serum 5 cells/ml, inoculated in 96-well plates, 100. Mu.l/well, at 37℃in 5% CO 2 Is cultured in an incubator. After 24 hours of cell wall-attached culture, the cells are changed into fresh culture solution, and meanwhile, the dosing treatment is carried out. The 3 compounds were combined with LPS at doses 1, 10, 30, 100. Mu.M. And a blank control is set. The final LPS concentration in each of the dosing groups was 100ng/ml. After the cells were dosed, the culture was continued for 24 hours, then MTT solution (10. Mu.L/well) was added to the cell solution, the cells were incubated with 0.25mg/ml MTT at 37℃for 3 hours, the culture solution was aspirated, and then 150. Mu.L of DMSO solution was added to determine the OD value of the optical density. And (3) data processing, namely performing data processing by using corresponding software of an enzyme-labeled instrument, calculating the average value of OD values of 3 holes of each sample, and calculating the cell viability (CV%) by using the average value according to the following formula.
Cell viability% = mean value of sample group OD value/mean value of blank group OD value x 100%
(5) Statistical method
All data were checked using the SPSS (27.0) statistical software package. Results were expressed as mean ± standard error, the difference in integrity was evaluated, the mean between groups was analyzed for homogeneity of variance using One-Way ANOVA analysis, and the comparison between groups was performed in combination with Dunnett's test analysis. The multisample variance homogeneity test uses a level test, when p >0.05, the variance is homogeneous, the difference in mean between the multiple groups is tested using Dunnett's double sided T, when p <0.05, the variance is heterogeneous, the difference in mean between the multiple groups is tested using Dunnett T3.
⑥IC 50 Is calculated by the method of (a)
Calculating IC by non-linear regression fitting of parameters such as each dose and inhibition rate 50 . Experimental results: see table 4.
TABLE 4 inhibition of LPS-induced release of NO by BV2 cells by alkaloid 1-11 results
Experimental results show that the lotus alkane alkaloid compounds 3,8 and 10 prepared from the fecal dustpan can obviously inhibit the NO release of the overactivated BV2 cells induced by LPS under the concentration which does not influence the survival rate of microglial BV2, thereby playing a role in preventing and treating neuroinflammation.

Claims (7)

1. The lotus alkyl alkaloid compound is characterized by having one of the following chemical structural formulas:
2.the method for preparing the lotus flower alkyl alkaloid compound as defined in claim 1, which is characterized in that: the method comprises the following process steps:
extracting whole plants of the fecal Vaccinium uliginosum (Stephania longa) by ethanol aqueous solution, and recovering the extracting solution to obtain ethanol extract; dissolving the ethanol extract with water, adjusting pH to 2, adding petroleum ether to obtain extract A, adjusting pH of water layer to 8, and adding dichloromethane to obtain extract B; separating the obtained extract B by silica gel column chromatography, and gradient eluting with mixed solvent I; separating the obtained fraction by ODS chromatography, and gradient eluting with mixed solvent II as mobile phase; the obtained eluent is further separated by preparative HPLC-UV, and the compound 10 is obtained by gradient elution with mixed solvent of methanol and water in a ratio of 4:6-9:1 or with mixed solvent of acetonitrile and water in a ratio of 2:8-8:2 as mobile phase,
the mixed solvent I is a mixed solvent formed by petroleum ether and ethyl acetate according to the volume ratio of 100:10-100:15, or is dichloromethane and methanol according to the volume ratio of 100: 3-100: 7, or a mixed solvent of chloroform and methanol with the volume ratio of 100: 3-100: 7, a mixed solvent; the mixed solvent II is methanol and water 1:9 to 9:1 mixed solvent, acetonitrile and water 1: 9-8: 2 mixing the solvents.
3.The method according to claim 2, characterized in that: the whole plant of the fecal sewage is heated and refluxed or cold soaked for 3 to 6 times by ethanol aqueous solution to obtain ethanol extract, wherein the volume concentration of the ethanol is 60 to 100 percent, and the fecal sewage is: the weight-volume ratio of the ethanol is 1:8-1:15 g/mL.
4. The method according to claim 2, characterized in that: after dissolving an ethanol extract of the whole plant of the fecal vaccinium uliginosum in water, adjusting the pH to 2 with 4.5% HCl, and adding petroleum ether to obtain an extract A; further adding 5% Na to the aqueous layer 2 CO 3 Adjusting pH to 8, and adding dichloromethane to obtain extract B; each solvent is extracted for 3 to 6 times, and the volume ratio of the water phase to the organic phase is 1:1.
5. The method according to claim 2, characterized in that: the mixed solvent II is methanol and water in a ratio of 3:7-8: 2, mixing the solvent or acetonitrile and water in a ratio of 1:9-6: 4, mixing the solvents; the mobile phase of the preparative HPLC-UV chromatography is methanol and water 4: 6-8: 2, mixing the solvents; or acetonitrile and water 3: 7-7: 3 mixing the solvents.
6. A pharmaceutical composition comprising the lotus-alkane-type alkaloid compound of claim 1.
7. Use of the lotus-alkane-type alkaloid compound of claim 1 or the pharmaceutical composition of claim 6 in the preparation of a medicament for preventing or treating neurodegenerative diseases.
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