Compound with anti-inflammatory effect in pyrethrum cinerariifolium root as well as preparation and application of compound

Abstract

The invention discloses an alkaloid compound extracted from anacyclus pyrethrum (L.) DC. As well as an extraction method and application thereof. The alkaloid compound is extracted from the anacyclus pyrethrum (L.) DC. Root (L.) by adopting an organic solvent, and the extraction method comprises the following steps: extracting the alkaloid compound from the anacyclus pyrethrum (L.) DC. And separating by using two, three or four combined methods of normal-phase silica gel column chromatography, reverse-phase silica gel column chromatography and semi-preparative high performance liquid chromatography to obtain the novel skeleton alkaloid compound. In addition, the compounds provided by the invention can play an anti-inflammatory role by inhibiting nitric oxide (NO) of cells, so that the compounds can be used for preparing anti-inflammatory drugs.

Description

[0001] This application is a divisional application of the Chinese invention patent application with the application date of August 30, 2023, application number 202311104612.0 and invention name "Compounds with anti-inflammatory effects in anacardia roots and their preparation and application". Technical Field

[0002] The present invention belongs to the field of medical technology, and specifically relates to compounds with anti-inflammatory effects in the root of Anabaena (compounds of Formula 1, Formula 2, Formula 3 or Formula 4) and preparation and application thereof. Background Art

[0003] Exploring lead compounds with significant pharmacological activity from traditional medicinal plants is a hot topic in drug research. Therefore, discovering novel compounds with anti-inflammatory effects from medicinal plants is an effective way to develop new anti-inflammatory drugs.

[0004] Anachyra root is the dried root of Anacyclus pyrethrum (L.) DC., a perennial herb of the genus Anacyclus, family Anthemideae, family Asteraceae. Other uses in traditional medicine include pharyngitis, tonsillitis, paralysis, epilepsy, fever, and diabetes. It has potent stimulant properties and is used as a salivator and tonic for the nervous system; a decoction of the root is used to treat tonsillitis, toothache, sore throat, and dental caries.

[0005] Modern pharmacological studies of anaqi root have shown that it has biological activities such as anti-epileptic, immunomodulatory, improvement of male sexual function, anti-mutation, anti-inflammatory, liver protection, hypoglycemic, and antibacterial. Studies have reported that it has N-alkylamide and piperidine alkaloids, but the correlation between these compounds and the pharmacological activity of anaqi root has not been fully clarified, and the material basis of the efficacy of anaqi root has not yet been elucidated. Summary of the invention

[0006] After intensive research, the inventors have isolated for the first time anti-inflammatory compounds (compounds of Formula 1, Formula 2, Formula 3 or Formula 4) with anti-inflammatory effects from the roots of Anacardiaceae. These compounds are new skeleton alkaloid compounds. Their structures have been identified, and cell experiments have verified that they have the effect of inhibiting the release of cellular nitric oxide (NO), and can be used to prepare anti-inflammatory drugs.

[0007] Therefore, the present invention provides the following aspects:

[0008] Aspect 1. A compound selected from the group consisting of:

[0009] N-(1-(2,2,5,5,8,12,12-heptamethyl-3,7-dione-1,2,3,4,5,6,7,8,9,11,12-decahydro-6H,13H-9a,14a-methanocyclopenta[7,8]azepino[4,5,6-ij]isoquinolin-2-yl)-2-methylpropyl-2-yl)acetamide (compound of formula 1 or compound 1);

[0010] 8a-acetyl-2,2,4,5a,7,7,10,10,11a-nonamethyl-2,5,5a,7,8,8a,10,11,11a,12-decahydropyrido[3',2':3,4]cyclopenta[1,2-g]pyrrolo[3,2-b]indolizin-10(1H)-one (compound of formula 2 or compound 2);

[0011] 2,2,6,6,8,8-hexamethyl-9a-(2-propionyl)-1,2,3,5,6,8,9,9a-octahydro-1H-pyrrolo[2,3-h]isoquinolin-1-one (compound of formula 3 or compound 3); and

[0012] 2-((5-acetyl-2,2,4-trimethyl-3,4-dihydro-2H-pyrrol-4-yl)methyl)-4,6,6-trimethylpyridin-3(6H)-one (compound of formula 4 or compound 4).

[0013] Aspect 2. The compound according to aspect 1 above or an isomer thereof, wherein the isomer is an enantiomer.

[0014] Aspect 3. The compound or isomer thereof according to Aspect 1 or 2 above, which is selected from Compound (+)-1, Compound (–)-1, Compound (+)-2, Compound (–)-2, Compound (+)-3, Compound (–)-3, Compound (+)-4 and Compound (–)-4, wherein:

[0015] Compound (+)-1 is: N-(1-((8R,9aS,10aS)-2,2,5,5,8,12,12-heptamethyl-3,7-dione-1,2,3,4,5,6,7,8,9,11,12-decahydro-6H,13H-9a,14a-methanocyclopenta[7,8]azepino[4,5,6-ij]isoquinolin-2-yl)-2-methylpropyl-2-yl)acetamide;

[0016] Compound (–)-1 is: N-(1-((8S,9aR,10aR)-2,2,5,5,8,12,12-heptamethyl-3,7-dione-1,2,3,4,5,6,7,8,9,11,12-decahydro-6H,13H-9a,14a-methanocyclopenta[7,8]azepino[4,5,6-ij]isoquinolin-2-yl)-2-methylpropyl-2-yl)acetamide;

[0017] Compound (+)-2 is: (5aR,8aR,11aR)-8a-acetyl-2,2,4,5a,7,7,10,10,11a-nonamethyl-2,5,5a,7,8,8a,10,11,11a,12-decahydropyrido[3',2':3,4]cyclopenta[1,2-g]pyrrolo[3,2-b]indolizin-10(1H)-one;

[0018] Compound (–)-2 is: (5aS,8aS,11aS)-8a-acetyl-2,2,4,5a,7,7,10,10,11a-nonamethyl-2,5,5a,7,8,8a,10,11,11a,12-decahydropyrido[3',2':3,4]cyclopenta[1,2-g]pyrrolo[3,2-b]indolizin-10(1H)-one;

[0019] Compound (+)-3 is: (S)-2,2,6,6,8,8-hexamethyl-9a-(2-propionyl)-1,2,3,5,6,8,9,9a-octahydro-1H-pyrrolo[2,3-h]isoquinolin-1-one;

[0020] Compound (–)-3 is: (R)-2,2,6,6,8,8-hexamethyl-9a-(2-propionyl)-1,2,3,5,6,8,9,9a-octahydro-1H-pyrrolo[2,3-h]isoquinolin-1-one;

[0021] Compound (+)-4 is: (R)-2-((5-acetyl-2,2,4-trimethyl-3,4-dihydro-2H-pyrrol-4-yl)methyl)-4,6,6-trimethylpyridin-3(6H)-one; and

[0022] Compound (–)-4 is: (S)-2-((5-acetyl-2,2,4-trimethyl-3,4-dihydro-2H-pyrrol-4-yl)methyl)-4,6,6-trimethylpyridin-3(6H)-one.

[0023] Aspect 4. The compound or isomer thereof according to any one of Aspects 1 to 3 above, which has a structural formula selected from the following:

[0024] .

[0025] Aspect 5. A method for extracting the compound or its isomer according to any one of aspects 1 to 4 above from the root of Anabaena, comprising the following steps:

[0026] a. Dry and crush the root of Anacardia, use 50-95% (v / v) ethanol aqueous solution, methanol or chloroform as solvent (ratio of medicinal material weight (kg) to solvent (L) 1:1.5-1:4), extract by cold soaking, percolation, heating reflux or ultrasonic extraction, and concentrate under reduced pressure to recover the solvent to obtain an extract;

[0027] b. After the total extract of step a is suspended in water, it is dispersed with an acid such as 1-5% hydrochloric acid or 1-5% sulfuric acid. The obtained acid-water layer is extracted with dichloromethane to remove non-alkaloids and then with an alkali such as NaHCO 3 、Na 2 CO 3 , adjusting the pH to about 10-12 with ammonia water or NaOH, extracting with an organic solvent such as dichloromethane, ethyl acetate or n-butanol, and concentrating under reduced pressure to recover the organic solvent to obtain total alkaloids; and

[0028] c. Separating the total alkaloids in step b by silica gel column chromatography, thin layer chromatography, reverse phase MCI column chromatography, Sephadex LH-20 column chromatography, high performance liquid chromatography or any combination thereof to obtain the compound or its isomers.

[0029] Aspect 6. The method according to Aspect 5 above, wherein in step c, separation is performed using a combination of normal phase silica gel column chromatography and one selected from reverse phase silica gel or reverse phase MCI column chromatography or semi-preparative high performance liquid chromatography. Preferably, after gradient or isocratic elution using normal phase silica gel column chromatography, reverse phase silica gel or reverse phase MCI column chromatography or semi-preparative high performance liquid chromatography is used to obtain a compound of Formula 1, Formula 2, Formula 3 or Formula 4. More preferably, the eluent used in the normal phase silica gel column chromatography is petroleum ether and ethyl acetate in a volume ratio of 100:0 to 3:1, dichloromethane and methanol in a volume ratio of 500:1 to 3:1, or petroleum ether in a volume ratio of 50:1 to 0:1. Oily ether and acetone, or methanol and water in a volume ratio of 1:9-1:0, the eluent used in the reversed-phase silica gel or reversed-phase MCI column chromatography is a methanol aqueous solution in a volume ratio of 10-100% (v / v) or an acetonitrile aqueous solution in a volume ratio of 20-100% (v / v), and the eluent for isocratic or gradient elution used in the semi-preparative high performance liquid chromatography is n-hexane / EtOH in a volume ratio of 99-50%, n-hexane / 2-isopropanol in a volume ratio of 99-50%, n-hexane / 2-isopropanol / diethylamine in a volume ratio of 99:1:0.002 to 50:50:0.002, or a methanol aqueous solution in a concentration of 20-100% (v / v).

[0030] Aspect 7. The method according to Aspect 5 above, wherein in step c, a combination of normal phase silica gel column chromatography, reverse phase silica gel or reverse phase MCI column chromatography, and semi-preparative high performance liquid chromatography is used for separation. Preferably, after gradient or isocratic elution using normal phase silica gel column chromatography, gradient elution is performed using reverse phase silica gel or reverse phase MCI column chromatography, and then semi-preparative high performance liquid chromatography is used to obtain a compound of Formula 1, Formula 2, Formula 3 or Formula 4. More preferably, the eluent used in the normal phase silica gel column chromatography is petroleum ether and ethyl acetate, dichloromethane and methanol or trichloromethane in a volume ratio of 100:1 to 0:1. The eluent used in the reversed-phase silica gel or reversed-phase MCI column chromatography is a methanol aqueous solution with a volume ratio of 10-100% (v / v) or an acetonitrile aqueous solution with a volume ratio of 20-100% (v / v), and the eluent used in the isocratic or gradient elution in the semi-preparative high performance liquid chromatography is n-hexane / EtOH with a volume ratio of 99-50%, n-hexane / 2-isopropanol with a volume ratio of 99-50%, n-hexane / 2-isopropanol / diethylamine with a volume ratio of 99:1:0.002 to 50:50:0.002, or a methanol aqueous solution with a concentration of 10-100% (v / v).

[0031] Aspect 8. The method according to aspect 5 above, wherein in step c, a combination of normal phase silica gel column chromatography, Sephadex LH-20 column chromatography, reverse phase silica gel or reverse phase MCI column chromatography, and semi-preparative high performance liquid chromatography is used for separation. Preferably, after gradient or isocratic elution using normal phase silica gel column chromatography, Sephadex LH-20 column chromatography, gradient elution using reverse phase silica gel or reverse phase MCI column chromatography, and then semi-preparative high performance liquid chromatography is used to obtain a compound of formula 1, formula 2, formula 3 or formula 4. More preferably, the eluent used in the normal phase silica gel column chromatography is petroleum ether and ethyl acetate, dichloromethane and methanol, or chloroform and methanol in a volume ratio of 100:1 to 0:1, and the The Sephadex LH-20 column chromatography adopts methanol gradient or isocratic elution, the eluent used in the reversed-phase silica gel or reversed-phase MCI column chromatography is a methanol aqueous solution with a volume ratio of 10-100% (v / v) or an acetonitrile aqueous solution with a volume ratio of 20-100% (v / v), and the eluent for isocratic or gradient elution used in the semi-preparative high performance liquid chromatography is n-hexane / EtOH with a volume ratio of 99-50% (v / v), n-hexane / 2-isopropanol with a volume ratio of 99-50% (v / v), n-hexane / 2-isopropanol / diethylamine with a volume ratio of 99:1:0.002 to 50:50:0.002 (v / v), or a methanol aqueous solution with a concentration of 20-100% (v / v).

[0032] Aspect 9. The method according to any one of Aspects 5 to 8 above, wherein in step c, the silica gel column chromatography is normal pressure or pressurized column chromatography, and / or the filler used is normal phase silica gel or reverse phase silica gel; and / or preferably, the method further comprises the step of subjecting the obtained racemate (i.e., the compound of Formula 1, Formula 2, Formula 3 or Formula 4) to chiral separation through a chiral chromatographic column to obtain enantiomeric compounds.

[0033] Aspect 10. Use of the compound or its isomer according to any one of Aspects 1 to 4 above in the preparation of an anti-inflammatory drug, preferably, the compound or its isomer exerts its anti-inflammatory effect by inhibiting the release of cellular nitric oxide (NO).

[0034] In summary, the present invention provides an alkaloid compound isolated from the root of Anacyclus, a preparation method and a use thereof, using Anacyclus pyrethrum (L.) DC. as a raw material, using solvent extraction, acid-base treatment, solvent extraction, separation by two, three or four methods of silica gel column chromatography, preparative thin layer chromatography, dextran gel LH-20 column chromatography or high performance liquid chromatography (pHPLC), and thin layer chromatography or high performance liquid chromatography (HPLC) detection and analysis to obtain 4 new skeleton alkaloid compounds. The compounds were tested for in vitro anti-inflammatory activity, and the experimental results showed that the new skeleton alkaloid compound isolated from the root of Anacyclus had certain anti-inflammatory activity and could be used to prepare anti-inflammatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the X-ray single crystal diffraction pattern of compound 1;

[0036] Figure 2 is the X-ray single crystal diffraction pattern of compound 3;

[0037] Figure 3 is the X-ray single crystal diffraction pattern of compound 4;

[0038] Figure 4 is compound 1 1 H NMR spectra;

[0039] Figure 5 is compound 1 13 C NMR spectra;

[0040] Figure 6 It is compound 2 1 H NMR spectra;

[0041] Figure 7 It is compound 2 13 C NMR spectra;

[0042] Figure 8 is compound 3 1 H NMR spectra;

[0043] Fig. 9 is compound 3 13 C NMR spectra;

[0044] Fig.10 is compound 4 1 H NMR spectra;

[0045] Fig.11 is compound 4 13 C NMR spectrum. DETAILED DESCRIPTION

[0046] The purpose of the present invention is to provide a compound with anti-inflammatory effect, a separation and preparation method thereof, and its application value in preparing anti-inflammatory drugs.

[0047] According to the present invention, in a first aspect, a compound having an anti-inflammatory effect is provided, wherein the structural formula of the compound (respectively a compound of Formula 1, Formula 2, Formula 3 or Formula 4) is as follows:

[0048] .

[0049] Formula 1

[0050] .

[0051] Formula 2

[0052] .

[0053] Formula 3

[0054] .

[0055] Formula 4

[0056] in:

[0057] Compound (+)-1 is: N-(1-((8R,9aS,10aS)-2,2,5,5,8,12,12-heptamethyl-3,7-dione-1,2,3,4,5,6,7,8,9,11,12-decahydro-6H,13H-9a,14a-methanocyclopenta[7,8]azepina[4,5,6-ij]isoquinolin-2-yl)-2-methylpropyl-2-yl)acetamide (compound of Formula 1);

[0058] Compound (–)-1 is: N-(1-((8S,9aR,10aR)-2,2,5,5,8,12,12-heptamethyl-3,7-dione-1,2,3,4,5,6,7,8,9,11,12-decahydro-6H,13H-9a,14a-methanocyclopenta[7,8]azepina[4,5,6-ij]isoquinolin-2-yl)-2-methylpropyl-2-yl)acetamide (compound of Formula 1);

[0059] Compound (+)-2 is: (5aR,8aR,11aR)-8a-acetyl-2,2,4,5a,7,7,10,10,11a-nonamethyl-2,5,5a,7,8,8a,10,11,11a,12-decahydropyrido[3',2':3,4]cyclopenta[1,2-g]pyrrolo[3,2-b]indolizin-10(1H)-one (compound of formula 2);

[0060] Compound (–)-2 is: (5aS,8aS,11aS)-8a-acetyl-2,2,4,5a,7,7,10,10,11a-nonamethyl-2,5,5a,7,8,8a,10,11,11a,12-decahydropyrido[3',2':3,4]cyclopenta[1,2-g]pyrrolo[3,2-b]indolizin-10(1H)-one (compound of formula 2);

[0061] Compound (+)-3 is: (S)-2,2,6,6,8,8-hexamethyl-9a-(2-propionyl)-1,2,3,5,6,8,9,9a-octahydro-1H-pyrrolo[2,3-h]isoquinolin-1-one (compound of formula 3);

[0062] Compound (–)-3 is: (R)-2,2,6,6,8,8-hexamethyl-9a-(2-propionyl)-1,2,3,5,6,8,9,9a-octahydro-1H-pyrrolo[2,3-h]isoquinolin-1-one (compound of Formula 3);

[0063] Compound (+)-4 is: (R)-2-((5-acetyl-2,2,4-trimethyl-3,4-dihydro-2H-pyrrol-4-yl)methyl)-4,6,6-trimethylpyridin-3(6H)-one (compound of formula 4);

[0064] Compound (–)-4 is: (S)-2-((5-acetyl-2,2,4-trimethyl-3,4-dihydro-2H-pyrrol-4-yl)methyl)-4,6,6-trimethylpyridin-3(6H)-one (compound of formula 4).

[0065] The above-mentioned method for extracting and separating alkaloid compounds is carried out according to the following steps:

[0066] The root of Anacardia is dried and crushed, and a 50-95% (v / v) ethanol aqueous solution, methanol or chloroform is used as a solvent (the ratio of the weight of the medicinal material (kg) to the solvent (L) is 1:1.5-1:4), and the extract is extracted by cold soaking, percolation, heating reflux or ultrasonic extraction, and the solvent is recovered by vacuum concentration to obtain an extract;

[0067] b. After the total extract of step a is suspended in water, it is dispersed with an acid such as 1-5% hydrochloric acid or 1-5% sulfuric acid. The obtained acid-water layer is extracted with dichloromethane to remove non-alkaloids and then with an alkali such as NaHCO 3 、Na 2 CO 3 , ammonia water or NaOH to adjust the pH to 10-12, then extract with an organic solvent such as dichloromethane, ethyl acetate or n-butanol, and concentrate under reduced pressure to recover the organic solvent to obtain total alkaloids;

[0068] c. Separating the total alkaloids in step b by two, three or four of the following methods: silica gel column chromatography, thin layer chromatography, reverse phase MCI column chromatography, Sephadex LH-20 column chromatography, and high performance liquid chromatography;

[0069] There are two separation methods:

[0070] The normal phase silica gel column chromatography eluent used is petroleum ether-ethyl acetate in a volume ratio of 100:0-3:1, dichloromethane-methanol in a volume ratio of 500:1-3:1, or petroleum ether / acetone in a volume ratio of 50:1-0:1 for gradient elution, and then reverse phase silica gel or MCI column chromatography or semi-preparative high performance liquid chromatography is used to obtain a compound of formula 1, formula 2, formula 3 or formula 4;

[0071] Three separation methods:

[0072] The normal phase silica gel column chromatography eluent is petroleum ether-ethyl acetate, dichloromethane-methanol or chloroform-methanol with a volume ratio of 100:1-0:1 for gradient elution, and then the compound is subjected to reverse phase silica gel or MCI column chromatography with a volume ratio (v / v) methanol aqueous solution or a 20-100% (v / v) acetonitrile aqueous solution gradient elution. Semi-preparative high performance liquid chromatography is performed with a volume ratio of 99-50% (v / v) n-hexane / EtOH, a volume ratio of 99-50% (v / v) n-hexane / 2-isopropanol or a volume ratio of 99:1:0.002-50:50:0.002 (v / v) n-hexane / 2-isopropanol / diethylamine as the eluent to obtain a compound of Formula 1, Formula 2, Formula 3 or Formula 4.

[0073] Four separation methods:

[0074] The normal phase silica gel column chromatography eluent is petroleum ether-ethyl acetate, dichloromethane-methanol or chloroform-methanol with a volume ratio of 100:1-0:1 (v / v) for gradient elution, and then the compound is subjected to Sephadex LH-20 column chromatography and isocratic elution with methanol. The compound is then subjected to reverse phase silica gel or MCI column chromatography and gradient elution with a volume ratio (v / v) of methanol aqueous solution or a 20-100% (v / v) acetonitrile aqueous solution. Semi-preparative high performance liquid chromatography is performed using a volume ratio of 99-50% (v / v) of n-hexane / EtOH, a volume ratio of 99-50% (v / v) of n-hexane / 2-isopropanol or a volume ratio of 99:1:0.002-50:50:0.002 (v / v / v) of n-hexane / 2-isopropanol / diethylamine as the eluent to obtain the compound of Formula 1, Formula 2, Formula 3 or Formula 4.

[0075] The method for preparing alkaloid compounds in the root of Anacardia described in step c is characterized in that the silica gel column chromatography used is normal pressure or pressurized column chromatography, the filler used is normal silica gel or reversed phase silica gel, and dichloromethane and methanol in a volume ratio of 500:1-3:1 (v / v); petroleum ether and ethyl acetate in a volume ratio of 1:0-3:1 (v / v); or methanol water in a volume ratio of 1:9-1:0 (v / v) is used as the eluent, and isocratic or gradient elution is adopted.

[0076] The method for preparing alkaloid compounds in the root of Anacardia described in step c is characterized in that the eluent of the Sephadex LH-20 column chromatography method is methanol, and isocratic elution is adopted.

[0077] The method for preparing alkaloid compounds in the root of Anachia described in step c is characterized in that the eluent of the preparative high performance liquid chromatography is a methanol aqueous solution with a volume ratio of 10-100% (v / v), n-hexane / EtOH with a volume ratio of 99-50% (v / v), n-hexane / 2-isopropanol with a volume ratio of 99-50% (v / v), or n-hexane / 2-isopropanol / diethylamine with a volume ratio of 99:1:0.002-50:50:0.002 (v / v / v), and isocratic or gradient elution is adopted.

[0078] According to another aspect of the present invention, a method for extracting and separating compounds with anti-inflammatory effects is provided, wherein the root of Anacardia is dried and then crushed, and a solvent is used as a solvent with a volume ratio of 50-95% ethanol aqueous solution, methanol or chloroform (the ratio of medicinal material weight (kg) to solvent (L) is 1:1.5-1:4), and cold immersion, percolation extraction, heating reflux or ultrasonic extraction are adopted, and the solvent is recovered by reduced pressure concentration to obtain an extract; the total extract is suspended in water, and then dispersed with an acid such as 1-5% hydrochloric acid or 1-5% sulfuric acid, and the obtained acid water layer is extracted with dichloromethane to remove non-alkaloids, and then with NaHCO 3 、Na2 CO 3 , ammonia water or NaOH to adjust the pH to 10-12, then extract with organic solvents such as chloroform, ethyl acetate or n-butanol, and concentrate under reduced pressure to recover the organic solvent to obtain total alkaloids; separate the obtained total alkaloids by two, three or four methods of silica gel column chromatography, thin layer chromatography, dextran gel LH-20 column chromatography or high performance liquid chromatography; and detect and analyze by thin layer chromatography or high performance liquid chromatography to obtain 8 new skeleton alkaloid compounds. The silica gel column chromatography used is normal pressure or pressurized column chromatography, the filler used is normal silica gel or reversed phase silica gel, and dichloromethane and methanol in a volume ratio of 500:1-3:1; petroleum ether and ethyl acetate in a volume ratio of 1:0-3:1; or methanol water in a volume ratio of 10:90-100:0 is used as the eluent, and isocratic or gradient elution is adopted. The preparative thin layer chromatography method used is normal pressure chromatography, and the developing system is a mixture of dichloromethane and methanol, a mixture of petroleum ether and acetone. The eluent of the Sephadex LH-20 column chromatography method used is chloroform-methanol or methanol with a volume ratio of 10:1, and isocratic elution is adopted. The eluent of the preparative high performance liquid chromatography method used is n-hexane / EtOH with a volume ratio of 99-50% (v / v), n-hexane / 2-propanol with a volume ratio of 99-50% (v / v), n-hexane / 2-isopropanol / diethylamine with a volume ratio of 99:1:0.002 to 50:50:0.002 (v / v), or a methanol aqueous solution with a concentration of 20-100% (v / v), and isocratic or gradient elution is adopted.

[0079] According to another aspect of the present invention, there is provided use of the compound having anti-inflammatory effect for preparing anti-inflammatory drugs.

[0080] The novel skeleton alkaloid compounds of the present invention were analyzed by combining a variety of spectral analysis methods (high-resolution mass spectrometry, ultraviolet spectroscopy, infrared spectroscopy, and nuclear magnetic resonance spectroscopy) and X-ray single crystal diffraction to determine the structures of compounds 1 to 4 prepared in the examples. The relative configurations of compound 1, compound 3, and compound 4 were determined by X-ray single crystal diffraction, such as Figure 1 , Figure 2 and Figure 3 shown.

[0081] Compound 1 (Anacyphrethine C): yellow block crystals; optical rotation value [α]25 D 128 (c 0.092, methanol, enantiomer compound (+)-1); [α]25 D -128 (c 0.092, methanol, enantiomer compound (-)-1); UV (methanol) λ max(log ε) 288 (3.92) nm, 417 (3.74) nm; IR (KBr) max 3291, 2973,2936, 2871, 1714, 1667,1563 and 1178 cm -1 ;ECD (c 1.86×10 -3 M, methanol) λ max 215 (-1.17), 289 (2.22), 337 (-1.97), 435 (1.67), enantiomer compound (+)-1; ECD (c 1.86×10 -3 M, methanol) λ max (Δε) 219 (1.43), 285 (-2.15), 377 (2.17), 432 (-1.74) nm, enantiomer compound (-)-1; high resolution mass spectrum m / z 496.3532 [M + H] + (Calculated value C 30 H 45 O 3 N 3 + ,496.3534). 1 H and 13 C NMR spectrum data are shown in Table 1.

[0082] Table 1 Compound 1 1 H and 13 C NMR data (deuterated chloroform, 600 MHz)

[0083]

[0084] Compound 2 (Anacyphrethine D): yellow powder; optical rotation value [α]25 D 92 (c 0.08, methanol, enantiomer compound (+)-2); [α]25 D -92 (c 0.066, methanol, enantiomer compound (-)-2); UV (methanol) λ max (log ε) 268 (408) nm, 453 (4.14) nm; IR (KBr) max 3342, 2970,2928, 2870, 1713, 1571,1365 and 1052 cm -1 ;ECD (c 1.83×10 -3 M, methanol) λ max(Δε) 216 (1.32), 243 (0.75), 274 (-2.85), 306 (0.77), 381 (-1.10), 446 (1.12), enantiomeric compound (+)-2; ECD (c 1.51×10 -3 M, methanol) λ max (Δε) 219 (-1.52), 243 (-1.44), 275 (5.01), 307 (-1.09), 383 (1.80), 446 (-1.71), enantiomer compound (-)-2; high resolution mass spectrum m / z 438.3114 [M + H] + (Calculated value C 27 H 40 O 2 N 3 + , 438.3115). 1 H and 13 The CNMR spectrum data are shown in Table 2.

[0085] Table 2 Compound 2 1 H and 13 C NMR data (deuterated chloroform, 600 MHz)

[0086]

[0087] Compound 3 (Anacyphrethine E): colorless block crystals; optical rotation value [α]25 D -34 (c 0.2, methanol, enantiomer compound (-)-3); [α]25 D 34 (c 0.2, methanol, enantiomer compound (+)-3); UV (methanol) λ max (log ε) 260 (4.22) nm; IR (KBr) max 2960, 2920, 1713, 1665, 1626,1399, 1355 and 1162 cm -1 ; ECD (c 1.21×10 -3 M, methanol) λ max (Δε) 218 ​​(0. 60), 257(-4.05), 288 (4.78) nm, enantiomer compound (-)-3; ECD (c 1.21×10 -3 M, methanol) λ max(Δε) 216 (-0.54), 260 (4.39), 288 (-4.86) nm, enantiomeric compound (+)-3; high resolution mass spectrum m / z 331.2376 [M + H] + (Calculated value C 20 H 31 O 2 N 2 + , 331.2380). 1 H and 13 C NMR spectrum data are shown in Table 3.

[0088] Compound 4 (Anacyphrethine F): colorless block crystals; optical rotation value [α]25 D 66 (c 0.2, methanol, enantiomer compound (+)-4); [α]25 D -66 (c 0.2, methanol, enantiomer compound (-)-4); UV (methanol) λ max (log ε) 253 (3.86) nm; IR (KBr) max 2970, 2927, 2866, 1697, 1662,1618, 1466, 1448 and 1358 cm -1 ;ECD (c 6.62×10 -4 M, methanol) λ max (Δε) 216 (22.06), 240 (-4.91), 267 (2.98), 336 (-1.81) nm, enantiomer compound (+)-4; ECD (c6.62×10 -4 M, methanol) λ max (Δε) 216 (-22.20), 241 (4.41), 272 (-2.91), 337 (2.28) nm, enantiomeric compound (-)-4; high resolution mass spectrum m / z 303.2063 [M + H] + (Calculated value C 18 H 27 O 2 N 2 + , 303.2067). 1 H and 13 C NMR spectrum data are shown in Table 3.

[0089] Table 3 Compounds 3 and 4 1 H and 13 C NMR data (deuterated chloroform, 600 MHz)

[0090]

[0091] In general, the above technical solution conceived by the present invention has the following technical advantages compared with the prior art:

[0092] (1) Compounds 1 to 4 provided in the present invention are novel skeleton compounds. Compound 1 is a pair of highly conjugated diamino 6 / 6 / 6 / 6 / 5 pentacyclic alkaloid novel skeleton enantiomers, with a unique 6,13-diazapentacyclic [10.3.3 1,3 .1.0 2, 7 .0 8,12 ] The skeleton structure of the nonadecane ring system has three discontinuous chiral stereocenters. Compound 2 is a pair of triamino 6 / 5 / 6 / 5 / 5 pentacyclic alkaloids with a new skeleton enantiomer, a unique 4,6,17-triazapentacyclic [10.7.0.0 4,11 .0 5,9 .0 13,18 ] The skeleton structure of the nonadecane ring system has three discontinuous chiral stereocenters. Compound 3 is a pair of triamino 6 / 6 / 5 tricyclic alkaloids with a new skeleton enantiomer, a unique 5,12-diazatricyclic [7.4.0 2,6 The skeleton structure of the nonadecane ring system has three discontinuous chiral stereo centers. Compound 4 is a pair of new pyridone-pyrrole skeleton enantiomers with one chiral stereo center.

[0093] (2) Compounds 1 to 4 provided in the present invention have anti-inflammatory activity and are useful for preparing anti-inflammatory drugs.

[0094] The present invention is further described in detail below with reference to the accompanying drawings and examples, but the scope of the present invention is not limited thereby. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the method, steps, conditions, etc. of the present invention all belong to the scope of the present invention.

[0095] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0096] The following are specific embodiments:

[0097] Example 1

[0098] Dried Anacyclus pyrethrum (L.) DC., 15.0 kg, was crushed and ultrasonically extracted with chloroform (30 L). The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, acidified with 5% hydrochloric acid, and then extracted with dichloromethane to remove non-alkaloid impurities. The acid aqueous solution was stirred in an ice-water bath and heated with saturated NaHCO 3 The aqueous solution is adjusted to pH 10 to obtain an alkalized solution, the alkalized solution is fully extracted with dichloromethane, and the dichloromethane extracts are combined and dried to obtain total alkaloids.

[0099] Example 2

[0100] Dried Anacyclus pyrethrum (L.) DC., 15.0 kg, was crushed, extracted by 95% ethanol percolation, and the extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, acidified with 5% hydrochloric acid, and then extracted with dichloromethane to remove non-alkaloid impurities. The acid aqueous solution was placed in an ice water bath and stirred continuously, and the pH was adjusted to 10 with an ammonia solution to obtain an alkaline solution. The alkaline solution was fully extracted with ethyl acetate, and the ethyl acetate extracts were combined and dried to obtain total alkaloids.

[0101] Example 3

[0102] Dried Anacyclus pyrethrum (L.) DC., 15.0 kg, was crushed and extracted with 50% ethanol (40 L) by percolation. The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, acidified with 2% hydrochloric acid, and then extracted with dichloromethane to remove non-alkaloid impurities. The acid solution was stirred in an ice-water bath with Na 2 CO 3 The aqueous solution is adjusted to pH 10 to obtain an alkalized solution. The alkalized solution is fully extracted with n-butanol, and the n-butanol extracts are combined and dried to obtain total alkaloids.

[0103] Example 4

[0104] Dried Anacyclus pyrethrum (L.) DC., 15.0 kg, was crushed, extracted with 75% ethanol (45 L) by reflux, and the extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, acidified with 1% hydrochloric acid, and then extracted with dichloromethane to remove non-alkaloid impurities. The acid aqueous solution was placed in an ice water bath and stirred continuously with a NaOH aqueous solution to adjust the pH to 12 to obtain an alkaline solution. The alkaline solution was fully extracted with dichloromethane, and the dichloromethane extracts were combined and dried to obtain total alkaloids.

[0105] Example 5

[0106] Dried Anacyclus pyrethrum (L.) DC., 15.0 kg, was crushed and extracted with methanol at room temperature. The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, acidified with 5% hydrochloric acid, and then extracted with dichloromethane to remove non-alkaloid impurities. The acid aqueous solution was stirred in an ice water bath with saturated NaHCO 3 The aqueous solution is adjusted to pH 10 to obtain an alkalized solution, the alkalized solution is fully extracted with dichloromethane, and the dichloromethane extracts are combined and dried to obtain total alkaloids.

[0107] Example 6

[0108] Dried Anacyclus pyrethrum (L.) DC., 15.0 kg, was crushed and extracted with 22.5 L methanol at room temperature. The extracts were concentrated under reduced pressure to obtain a total extract. The total extract was suspended in water, acidified with 1% sulfuric acid, and then extracted with dichloromethane to remove non-alkaloid impurities. The acid solution was stirred in an ice water bath with saturated NaHCO 3 The aqueous solution is adjusted to pH 10 to obtain an alkalized solution, the alkalized solution is fully extracted with dichloromethane, and the dichloromethane extracts are combined and dried to obtain total alkaloids.

[0109] Example 7

[0110] Dried Anacyclus pyrethrum (L.) DC., 15.0 kg, was crushed and extracted with 22.5 L methanol at room temperature. The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, acidified with 2% sulfuric acid, and then extracted with dichloromethane to remove non-alkaloid impurities. The acid aqueous solution was stirred in an ice water bath with saturated NaHCO 3 The aqueous solution is adjusted to pH 10 to obtain an alkalized solution, the alkalized solution is fully extracted with dichloromethane, and the dichloromethane extracts are combined and dried to obtain total alkaloids.

[0111] Example 8

[0112] Dried Anacyclus pyrethrum (L.) DC., 15.0 kg, was crushed and extracted with 22.5 L methanol at room temperature. The extracts were concentrated under reduced pressure and combined to obtain a total extract. The total extract was suspended in water, acidified with 5% sulfuric acid, and then extracted with dichloromethane to remove non-alkaloid impurities. The acid aqueous solution was stirred in an ice water bath with saturated NaHCO 3 The aqueous solution is adjusted to pH 10 to obtain an alkalized solution, the alkalized solution is fully extracted with dichloromethane, and the dichloromethane extracts are combined and dried to obtain total alkaloids.

[0113] Example 9

[0114] Any of the total alkaloids in Examples 1-8 were mixed with 100-200 mesh silica gel, subjected to silica gel column chromatography, and then gradient eluted with dichloromethane / methanol (100:0-3:1, V / V), and the same components were combined to obtain 6 components with increasing polarity, namely, Fr.A, Fr.B, Fr.C, Fr.D, Fr.E and Fr.F; wherein the first component Fr.A (159.0 g) was mixed with 100-200 mesh silica gel, subjected to silica gel column chromatography, and then gradient eluted with petroleum ether / ethyl acetate (100:0-3:1, V / V), and the same components were combined to obtain 6 components with increasing polarity, namely, Fr.A1-Fr.A6; wherein the second component Fr.A2 (76.1 g) was subjected to reverse phase MCI column chromatography and gradient elution with acetonitrile / water (20:80-100:0, V / V). The same components were combined to obtain 7 subcomponents Fr.A21-Fr.A27 with decreasing polarity. Among them, component Fr.A25 (10.0 g) was mixed with 200-300 mesh silica gel, subjected to silica gel column chromatography, and then gradient eluted with petroleum ether / ethyl acetate (10:1-3:1, V / V). The same components were combined to obtain 3 subcomponents Fr.A251-Fr.A253 with decreasing polarity. Component Fr.A253 (9.0 g) The sample was mixed with 200-300 mesh silica gel and subjected to silica gel column chromatography, followed by gradient elution with petroleum ether / ethyl acetate (10:1-3:1, V / V). The same components were combined to obtain 6 components Fr.A2531-Fr.A2533 with increasing polarity. Among them, components Fr.A251 and Fr.A2531 (136.3 mg) were combined and separated by semi-preparative HPLC C18 column with methanol / water (10:100-100:0, V / V) as the mobile phase at a flow rate of 3 mL / min to obtain compound 4 (Anacyphrethines F, 45.0 mg, 0.000300%) with a retention time of t R The racemate of compound 4 was chirally separated by chiral chromatography on a chiral column (DAICEL CORPORATION Chiralpak ID 5 µm 10 × 250 mm; solvent: n-hexane / EtOH = 97:3; column temperature: 25 °C; flow rate: 3 ml / min; detection wavelength: 254 nm) to obtain enantiomer compound (+)-4 (13.0 mg, t R = 9.7 min) and enantiomer compound (-)-4 (13.3 mg, t R= 11.2 min). Component Fr.A5 (22.9 g) was subjected to reverse phase MCI column chromatography and eluted with methanol / water gradient (10:90-100:0, V / V). The same components were combined to obtain 9 sub-components Fr.A51-Fr.A59 with decreasing polarity. Component Fr.A58 (702.0 mg) was subjected to Sephadex LH-20 gel column chromatography and eluted with methanol to obtain 4 sub-components Fr.A581-Fr.A584 with decreasing molecular weight. Component Fr.A582 (120.0 mg) was subjected to semi-preparative high performance liquid chromatography C 18 Column separation, the mobile phase was methanol / water (70:30, V / V), the flow rate was 3 mL / min, and compound 2 (Anacyphrethines D, 15.0 mg, 0.000300%) was obtained with a retention time of t R The racemate of compound 2 was chirally separated by chiral chromatography on a chiral column (DAICELCORPORATION Chiralpak ID 5 µm 10 × 250 mm; solvent: n-hexane / EtOH = 95:5; column temperature: 25°C; flow rate: 3 ml / min; detection wavelength: 254 nm) to obtain enantiomer compound (+)-2 (6.5 mg, t R = 13.4 min) and enantiomer compound (-)-2 (6.3 mg, t R = 18.4 min).

[0115] Component Fr.B (179.1 g) was subjected to reverse phase MCI column chromatography and gradient elution with methanol / water (20:80-100:0, V / V). The same components were combined to obtain two sub-components Fr.B1-Fr.B2 with decreasing polarity. Component Fr.B1 (76.1 g) was subjected to silica gel column chromatography and then gradient elution with petroleum ether / ethyl acetate (1:0-3:1, V / V). The same components were combined to obtain four sub-components Fr.B1a-Fr.B1d with decreasing polarity. Component Fr.B1b (18.6 g) was subjected to reverse phase MCI column chromatography and gradient elution with methanol / water (10:90-100:0, V / V). The same components were combined to obtain six sub-components Fr.B1b1-Fr.B1b6 with decreasing polarity. Component Fr.B1b4 (9.6 The component Fr.B1b42 (2.0 g) was chromatographed on a normal phase silica gel column and eluted with dichloromethane / methanol (500:1-10:1, V / V) to obtain three subfractions Fr.B1b41-Fr.B1b44 with decreasing polarity. The component Fr.B1b42 (2.0 g) was chromatographed on a Sephadex LH-20 gel column and eluted with methanol to obtain two subfractions Fr.B1b42a-Fr.B1b42b with decreasing molecular weight. The component Fr.B1b42b (1.9 g) was chromatographed on a reverse phase C18 silica gel column and eluted with acetonitrile / water gradient (20:80-100:0, V / V). The same components were combined to obtain 11 subfractions Fr.B1b42b1-Fr.B1b42b11 with decreasing polarity. The component Fr.B1b42b1 (552.0 The fraction Fr.B1b42b1c (183.2 mg) was chromatographed on a normal phase silica gel column and eluted with dichloromethane / methanol (500:1-5:1, V / V) to obtain five subfractions Fr.B1b42b1a-Fr.B1b42b1e with decreasing polarity. The fraction Fr.B1b42b1c (183.2 mg) was chromatographed on a Sephadex LH-20 gel column and eluted with methanol to obtain four subfractions Fr.B1b42b1c1-Fr.B1b42b1c4 with decreasing molecular weight. The fraction Fr.B1b42b1c2 (127.9 mg) was chromatographed on a reverse phase C 18 Silica gel column chromatography was performed with a methanol / water gradient elution (10:90-100:0, V / V). The same components were combined to obtain four subcomponents Fr.B1b42b1c2a-Fr.B1b42b1c2d with decreasing polarity. Component Fr.B1b42b1c2d (15.5 mg) was subjected to semi-preparative HPLC C 18 Column separation, the mobile phase was methanol / water (36:64, V / V), the flow rate was 3 mL / min, and compound 3 (Anacyphrethines C, 17.7 mg, 0.000113%) was obtained; retention time t RThe racemate of compound 3 was chirally separated by chiral chromatography on a chiral column (DAICEL CORPORATION Chiralpak ID 5 µm 10 × 250 mm; solvent: n-hexane / 2-propanol = 85:15; column temperature: 25 °C; flow rate: 3 ml / min; detection wavelength: 254 nm) to obtain enantiomer compound (+)-3 (1.5 mg, t R = 17.6 min) and enantiomer compound (-)-3 (1.4 mg, t R = 12.6 min). Component Fr.B1b5 (2.6 g) was chromatographed on a normal phase silica gel column and eluted with dichloromethane / methanol (500:1~5:1, V / V) to obtain three sub-components Fr.B1b51−Fr.B1b53 with decreasing polarity. Component Fr.B1b52 (983.0 mg) was chromatographed on a Sephadex LH-20 gel column and eluted with methanol to obtain two sub-components Fr.B1b52a−Fr.B1b52b with decreasing molecular weight. Component Fr.B1b52b (736.9 The fraction Fr.B1b52b5 (175.0 mg) was chromatographed on a reverse phase C18 silica gel column and eluted with a methanol / water gradient (20:80-100:0, V / V). The same fractions were combined to obtain 6 subfractions Fr.B1b52b1-Fr.B1b52b6 with decreasing polarity. The fraction Fr.B1b52b5 (175.0 mg) was chromatographed on a normal phase silica gel column and eluted with dichloromethane / methanol (500:1-5:1, V / V) to obtain 3 subfractions Fr.B1b52b5a-Fr.B1b52b5c with decreasing polarity. The fraction Fr.B1b52b5b (170.0 mg) was chromatographed on a Sephadex column and eluted with dichloromethane / methanol (500:1-5:1, V / V). LH-20 gel column chromatography, eluted with methanol, obtained three subfractions Fr.B1b52b5b1-Fr.B1b52b5b3 with decreasing molecular weight; component Fr.B1b52b5b2 (123.0 mg) was chromatographed on a normal phase silica gel column, eluted with dichloromethane / methanol (300:1-5:1, V / V), obtained two subfractions Fr.B1b52b5b2a-Fr.B1b52b5b2b with decreasing polarity; component Fr. B1b52b5b2a (92.5 mg) was chromatographed on a semi-preparative HPLC C 18 Column separation, mobile phase acetonitrile / water (20:100-100:0, V / V), flow rate 3 mL / min, to obtain compound 1 (Anacyphrethines C, 17.7 mg, 0.000113%); retention time t RThe racemate of compound 1 was chirally separated by chiral chromatography on a chiral column (DAICELCORPORATION Chiralpak ID 5 µm 10 × 250 mm; solvent: n-hexane / 2-propanol / diethylamine = 98:2:0.002; column temperature: 25 °C; flow rate: 3 ml / min; detection wavelength: 254 nm) to obtain enantiomer compound (+)-1 (6.2 mg, t R = 30.8 min) and enantiomer compound (-)-1 (6.0 mg, t R = 33.3 min).

[0116] Example 10

[0117] Any of the total alkaloids in the above total alkaloid examples 1-8 was mixed with 100-200 mesh silica gel, subjected to silica gel column chromatography, and then gradient eluted with dichloromethane / methanol (100:0-3:1, V / V), and the same components were combined to obtain 6 components Fr.A, Fr.B, Fr.C, Fr.D, Fr.E and Fr.F with increasing polarity; wherein the first component Fr.A (159.0 g) was repeatedly subjected to silica gel column chromatography, using petroleum ether-ethyl acetate with a volume ratio of 100:0-3:1, dichloromethane-methanol with a volume ratio of 500:1-3:1 or petroleum ether / acetone with a volume ratio of 50:1-0:1 as an eluent for gradient elution to obtain compound 4 (Anacyphrethines F, 45.0 mg, 0.000300%) and compound 2 (Anacyphrethines D, 15.0 The racemate of compound 4 was chirally resolved on a chiral chromatographic column (DAICEL CORPORATION Chiralpak ID 5 µm 10 × 250 mm; solvent: n-hexane / EtOH = 99:1-50-50; column temperature: 25 °C; flow rate: 3 ml / min; detection wavelength: 254 nm) to obtain enantiomer compound (+)-4 (13.0 mg, t R = 9.7 min) and the enantiomer compound (-)-4 (13.3 mg, t R = 11.2min). The racemic compound 2 was chirally separated by a chiral chromatographic column (DAICEL CORPORATION Chiralpak ID 5 µm 10 ×250 mm; solvent: n-hexane / EtOH = 99:1-50:50; column temperature: 25 °C; flow rate: 3 ml / min; detection wavelength: 254 nm) to obtain the enantiomer compound (+)-2 (6.5 mg, t R= 13.4 min) and enantiomer compound (-)-2 (6.3 mg, t R = 18.4 min).

[0118] Component Fr.B (179.1 g) was subjected to repeated silica gel column chromatography, using petroleum ether-ethyl acetate with a volume ratio of 100:0-3:1, dichloromethane-methanol with a volume ratio of 500:1-3:1 or petroleum ether / acetone with a volume ratio of 50:1-0:1 as the eluent for gradient elution to obtain compound 3 (Anacyphrethines C, 17.7 mg, 0.000113%) and compound 1 (Anacyphrethines C, 17.7 mg, 0.000113%); the racemate of compound 3 was subjected to chiral separation on a chiral chromatographic column (DAICEL CORPORATIONChiralpak ID 5 µm 10 × 250 mm; solvent: n-hexane / 2-isopropanol = 99:1-50:50; column temperature: 25 °C; flow rate: 3 ml / min; detection wavelength: 254 nm) to obtain enantiomer compound (+)-3 (1.5 mg, t R =17.6 min) and enantiomer compound (-)-3 (1.4 mg, t R = 12.6 min); the racemate of compound 1 was subjected to chiral separation on a chiral chromatographic column (DAICEL CORPORATION Chiralpak ID 5 µm 10 × 250 mm; solvent: n-hexane / 2-isopropanol / diethylamine = 99:1:0.002-50:50:0.002; column temperature: 25 °C; flow rate: 3 ml / min; detection wavelength: 254 nm) to obtain enantiomer compound (+)-1 (6.2 mg, t R = 30.8 min) and enantiomer compound (-)-1 (6.0 mg, t R = 33.3 min).

[0119] Embodiment 11

[0120] Anti-inflammatory activity of the novel skeleton alkaloid compounds isolated from the root of Anacardia ovata

[0121] 1. Cell culture:

[0122] Mouse macrophage Raw264.7 cells (purchased from BeNa Culture Collection, BNCC) were cultured in Dulbecco's modified eagle medium (DMEM) high glucose medium (purchased from Hyclone, USA) containing 10% fetal bovine serum (FBS) (purchased from Giboco, USA) and 1% penicillin and streptomycin at 37°C and 5% CO 2 Cultured in an incubator;

[0123] 2. Test of the effect of the compounds of the present invention on cell viability:

[0124] The compound of the present invention was dissolved in dimethyl sulfoxide (DMSO), and Raw264.7 in the logarithmic growth phase and in good growth condition was taken and 5 × 10 3 Each well was connected to a 96-well plate. Various compounds of different concentrations were added to the experimental group, and dimethyl sulfoxide (DMSO) was added to the control group. After culturing for 24 hours, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt (CCK-8 reagent) was added to each well. The absorbance at 450 nm was measured using an enzyme reader, and the cell survival rate was calculated. The experimental results are shown in Table 4.

[0125] Table 4. Cell viability of compounds 1-4

[0126]

[0127] 3. Determination of nitric oxide (NO) content:

[0128] The Griess method (Arias-Negrete et al., Analytical Biochemistry 328.1(2004):14-21) was used to test the release of NO in Raw264.7 cells. After adding the test monomer compound and incubating the cells for 1 hour, 1 μg / mL lipopolysaccharide (LPS, Sigma, L4391) was added and incubated for 22 hours. After the incubation was completed, the cell supernatant was collected and the content of nitric oxide in the cell supernatant was determined by the Griess method. Before the determination, GriessReagent I and II (Nitric Oxide Assay kit, Beyotine, S0021M) were taken out and restored to room temperature. The standard was diluted with complete culture medium (1-100 μM). The concentration of the standard can be 0, 1, 2, 5, 10, 20, 40, 60, 100 μM, add the standard and the collected culture supernatant to a 96-well plate at 50 μL / well, add 50 μL of Griess Reagent I and 50 μL of Griess Reagent II restored to room temperature to each well in turn, shake and mix for 5 min, measure the absorbance at 540 nm, make a standard curve, and calculate the NO content in the culture supernatant based on the standard curve; the initial screening concentration of the monomer compound is 40 μM, and the inhibition rate results are shown in Table 4.

[0129] Table 5. NO inhibitory activity of compounds 1-4

[0130]

[0131] [a] Positive drug: Andrograholide (AG, HY-N0191). [b] Each experiment was repeated 3 times.

[0132] Conclusion: Compounds 1 to 4 all have a certain inhibitory effect on the release of NO. The IC values ​​of compounds (+)-1, (–)-1, (+)-2, (–)-2, (+)-4 and (–)-4 are 50 The values ​​were 56.21±2.14, 73.13±3.78, 44.14±1.47 μM, 41.10±3.15 μM, 65.82±1.77 and 84.13±2.37, respectively. Compound (+)-2 and compound (–)-2 had a significant inhibitory effect on the release of NO.

[0133] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Alkaloid compounds extracted from anacardia root: 2-((5-acetyl-2,2,4-trimethyl-3,4-dihydro-2H-pyrrol-4-yl)methyl)-4,6,6-trimethylpyridin-3(6H)-one.

2. The alkaloid compound according to claim 1, which is selected from compound (+)-4 or compound (-)-4, in: Compound (+)-4 is: (R)-2-((5-acetyl-2,2,4-trimethyl-3,4-dihydro-2H-pyrrol-4-yl)methyl)-4,6,6-trimethylpyridin-3(6H)-one; Compound (–)-4 is: (S)-2-((5-acetyl-2,2,4-trimethyl-3,4-dihydro-2H-pyrrol-4-yl)methyl)-4,6,6-trimethylpyridin-3(6H)-one, They have the following structural formulas respectively: or .

3. A method for extracting the alkaloid compound according to claim 1 or 2 from the root of Anacardia, wherein The following steps are involved: a. Dry and crush the root of Anacardia, use 50-95% by volume of ethanol aqueous solution, methanol or chloroform as solvent, wherein the ratio of the weight of Anacardia root in kg to the volume of solvent in L is 1:1.5-1:4, extract by cold soaking, percolation, heating reflux or ultrasonic extraction, and concentrate under reduced pressure to recover the solvent to obtain an extract; b. Suspending the total extract of step a in water, dispersing it with acid, extracting the obtained acid-water layer with dichloromethane to remove non-alkaloids, adjusting the pH to 10-12 with alkali, extracting it with an organic solvent such as dichloromethane, ethyl acetate or n-butanol, and concentrating it under reduced pressure to recover the organic solvent to obtain total alkaloids; and c. Using normal phase silica gel column chromatography to perform gradient or isocratic elution on the total alkaloids in step b, and then separating them using one selected from reverse phase silica gel column chromatography, reverse phase MCI column chromatography and semi-preparative high performance liquid chromatography to obtain the alkaloid compound, wherein the eluent used in the normal phase silica gel column chromatography is petroleum ether and ethyl acetate in a volume ratio of 100:0 to 3:1, dichloromethane and methanol in a volume ratio of 500:1 to 3:1, petroleum ether and acetone in a volume ratio of 50:1 to 0:1, or methanol and water in a volume ratio of 1:9-1:0, and the reverse The eluent used in the reverse phase silica gel or reverse phase MCI column chromatography is a methanol aqueous solution with a volume ratio of 10-100% or an acetonitrile aqueous solution with a volume ratio of 20-100%, and the eluent for isocratic or gradient elution used in the semi-preparative high performance liquid chromatography is n-hexane / EtOH with a volume ratio of 99-50%, n-hexane / 2-isopropanol with a volume ratio of 99-50%, n-hexane / 2-isopropanol / diethylamine with a volume ratio of 99:1:0.002 to 50:50:0.002, or a methanol aqueous solution with a volume ratio of 20-100%.

4. A method for extracting the alkaloid compound according to claim 1 or 2 from the root of Anacardia, wherein The following steps are involved: a. Dry and crush the root of Anacardia, use 50-95% by volume of ethanol aqueous solution, methanol or chloroform as solvent, wherein the ratio of the weight of Anacardia root in kg to the volume of solvent in L is 1:1.5-1:4, extract by cold soaking, percolation, heating reflux or ultrasonic extraction, and concentrate under reduced pressure to recover the solvent to obtain an extract; b. Suspending the total extract of step a in water, dispersing it with acid, extracting the obtained acid-water layer with dichloromethane to remove non-alkaloids, adjusting the pH to 10-12 with alkali, extracting it with an organic solvent such as dichloromethane, ethyl acetate or n-butanol, and concentrating it under reduced pressure to recover the organic solvent to obtain total alkaloids; and c. The total alkaloids in step b are subjected to gradient or isocratic elution using normal phase silica gel column chromatography, and then gradient eluted using reverse phase silica gel column chromatography or reverse phase MCI column chromatography, and then separated using semi-preparative high performance liquid chromatography to obtain the alkaloid compound, wherein the eluent used in the normal phase silica gel column chromatography is petroleum ether and ethyl acetate, dichloromethane and methanol, or chloroform and methanol in a volume ratio of 100:1 to 0:1, and the eluent used in the reverse phase silica gel or reverse phase MCI column chromatography is It is a methanol aqueous solution with a volume ratio of 10-100% or an acetonitrile aqueous solution with a volume ratio of 20-100%, and the eluent for isocratic or gradient elution used in the semi-preparative high performance liquid chromatography is n-hexane / EtOH with a volume ratio of 99-50%, n-hexane / 2-isopropanol with a volume ratio of 99-50%, n-hexane / 2-isopropanol / diethylamine with a volume ratio of 99:1:0.002 to 50:50:0.002, or a methanol aqueous solution with a volume ratio of 10-100%.

5. A method for extracting the alkaloid compound according to claim 1 or 2 from the root of Anacardia, wherein The following steps are involved: a. Dry and crush the root of Anacardia, use 50-95% by volume of ethanol aqueous solution, methanol or chloroform as solvent, wherein the ratio of the weight of Anacardia root in kg to the volume of solvent in L is 1:1.5-1:4, extract by cold soaking, percolation, heating reflux or ultrasonic extraction, and concentrate under reduced pressure to recover the solvent to obtain an extract; b. Suspending the total extract of step a in water, dispersing it with acid, extracting the obtained acid-water layer with dichloromethane to remove non-alkaloids, adjusting the pH to 10-12 with alkali, extracting it with an organic solvent such as dichloromethane, ethyl acetate or n-butanol, and concentrating it under reduced pressure to recover the organic solvent to obtain total alkaloids; and c. The total alkaloids in step b are subjected to gradient or isocratic elution using normal phase silica gel column chromatography, and then subjected to Sephadex LH-20 column chromatography, followed by gradient elution using reverse phase silica gel column chromatography or reverse phase MCI column chromatography, and finally separated by semi-preparative high performance liquid chromatography to obtain the alkaloid compound, wherein the eluent used in the normal phase silica gel column chromatography is petroleum ether and ethyl acetate, dichloromethane and methanol, or chloroform and methanol in a volume ratio of 100:1 to 0:1, wherein the eluent used in the normal phase silica gel column chromatography is petroleum ether and ethyl acetate, dichloromethane and methanol, or chloroform and methanol in a volume ratio of 100:1 to 0:

1. The semi-preparative high performance liquid chromatography method comprises the following steps: the eluent for isocratic or gradient elution is n-hexane / EtOH with a volume ratio of 99-50%, n-hexane / 2-isopropanol with a volume ratio of 99-50%, n-hexane / 2-isopropanol / diethylamine with a volume ratio of 99:1:0.002 to 50:50:0.002, or a methanol aqueous solution with a volume ratio of 20-100%.

6. The method according to any one of claims 3 to 5, wherein in step b, the acid used is hydrochloric acid with a concentration of 1-5% or sulfuric acid with a concentration of 1-5%.

7. The process according to any one of claims 3 to 5, wherein in step b, the base used is NaHCO 3 、Na 2 CO 3 , ammonia or NaOH.

8. The method according to any one of claims 3 to 5, wherein in step c, the silica gel column chromatography is normal pressure or pressurized column chromatography, and the filler used is normal phase silica gel or reverse phase silica gel.

9. The method according to any one of claims 3 to 5, wherein the method further comprises the step of subjecting the compound obtained as a racemate to chiral resolution through a chiral chromatography column to obtain enantiomeric compounds.

10. Use of the compound according to claim 1 or 2 in the preparation of anti-inflammatory drugs, wherein the compound exerts anti-inflammatory effects by inhibiting the release of cellular nitric oxide.

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