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 a Chinese patent application with an application date of August 30, 2023, an application number of 202311104612.0, and an invention title of "Compounds with anti-inflammatory effects in Anacyclus pyrethrum roots, their preparation and applications". Technical Field

[0002] The present invention belongs to the field of pharmaceutical technology, and specifically relates to compounds with anti-inflammatory effects in Anacyclus pyrethrum roots (compounds of Formula 1, Formula 2, Formula 3 or Formula 4), their preparation and applications. Background Art

[0003] Searching for lead compounds with significant pharmacodynamic activities 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] Anacyclus pyrethrum root is the dried root of Anacyclus pyrethrum (L.) DC., which is a perennial herb of the genus Anacyclus in the tribe Anthemideae of the family Asteraceae. Other uses in traditional medicine include pharyngitis, tonsillitis, paralysis, epilepsy, fever and diabetes. It has strong stimulating properties and can be used as a sialagogue and a tonic for the nervous system; the root decoction can be used to treat tonsillitis, toothache, sore throat and dental caries.

[0005] Anacyclus pyrethrum root is a classic and commonly used medicinal material in traditional Uyghur medicine and is widely used in Uyghur medicine finished preparations; modern pharmacological studies have shown that it has biological activities such as anti-epilepsy, immunomodulation, improvement of male sexual function, anti-mutation, anti-inflammatory, liver protection, hypoglycemic, antibacterial, etc. Research reports that it contains N-alkylamide and piperidine alkaloids, but the correlation between these compounds and the pharmacological activities of Anacyclus pyrethrum root has not been fully clarified, and the pharmacodynamic material basis of Anacyclus pyrethrum root has not been elucidated. Summary of the Invention

[0006] After painstaking research, the inventors of the present invention have first isolated anti-inflammatory compounds with anti-inflammatory effects (compounds of Formula 1, Formula 2, Formula 3 or Formula 4) from Anacyclus pyrethrum roots. These compounds are novel skeleton alkaloid compounds, their structures have been identified, and through cell experiments, it has been verified that they have an inhibitory effect on 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 or its isomer selected from the following:

[0009] N-(1-(2,2,5,5,8,12,12-heptamethyl-3,7-dioxo-1,2,3,4,5,6,7,8,9,11,12-decahydro-6H,13H-9a,14a-methanocyclopenta[7,8]azoni[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]indazol-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 or its isomer according to Aspect 1 above, wherein the isomer is an enantiomer.

[0014] Aspect 3. The compound or its isomer 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-dioxo-1,2,3,4,5,6,7,8,9,11,12-decahydro-6H,13H-9a,14a-methanocyclopenta[7,8]azoni[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-dioxo-1,2,3,4,5,6,7,8,9,11,12-decahydro-6H,13H-9a,14a-methano-cyclopenta[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]indazol-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]indazol-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 its isomer according to any one of the above Aspects 1 to 3, 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 Anacroot, comprising the following steps:

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

[0027] b. After suspending the total extract of step a in water, disperse and treat it with an acid such as hydrochloric acid with a concentration of 1 - 5% or sulfuric acid with a concentration of 1 - 5%. For the obtained acid aqueous layer, extract with dichloromethane to remove non - alkaloids, then adjust the pH to about 10 - 12 with an alkali such as NaHCO3, Na2CO3, ammonia water, or NaOH, and 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; and

[0028] c. Separate the total alkaloids of 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 isomer.

[0029] Aspect 6. The method according to Aspect 5 above, wherein in step c, normal - phase silica gel column chromatography and a combination of two selected from reverse - phase silica or reverse - phase MCI column chromatography or semi - preparative high - performance liquid chromatography are used for separation. Preferably, after gradient or isocratic elution using normal - phase silica gel column chromatography, reverse - phase silica or reverse - phase MCI column chromatography or semi - preparative high - performance liquid chromatography is used to obtain the 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 with a volume ratio of 100:0 to 3:1, dichloromethane and methanol with a volume ratio of 500:1 to 3:1, petroleum ether and acetone with a volume ratio of 50:1 to 0:1, or methanol and water with a volume ratio of 1:9 - 1:0. The eluent used in the reverse - phase silica or reverse - phase MCI column chromatography is an aqueous methanol solution with a volume ratio of 10 - 100% (v / v) or an aqueous acetonitrile 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%, 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 an aqueous methanol solution with 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 carried out using reverse-phase silica gel or reverse-phase MCI column chromatography, and then semi-preparative high-performance liquid chromatography is employed to obtain the 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, the eluent used in the reverse-phase silica gel or reverse-phase MCI column chromatography is an aqueous methanol solution in a volume ratio of 10-100% (v / v) or an aqueous acetonitrile 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-propanol in a volume ratio of 99-50%, n-hexane / 2-propanol / diethylamine in a volume ratio of 99:1:0.002 to 50:50:0.002, or an aqueous methanol 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 is carried out, gradient elution is carried out using reverse-phase silica gel or reverse-phase MCI column chromatography, and then semi-preparative high-performance liquid chromatography is employed to obtain the 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, the Sephadex LH-20 column chromatography uses methanol gradient or isocratic elution, the eluent used in the reverse-phase silica gel or reverse-phase MCI column chromatography is an aqueous methanol solution in a volume ratio of 10-100% (v / v) or an aqueous acetonitrile 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% (v / v), n-hexane / 2-propanol in a volume ratio of 99-50% (v / v), n-hexane / 2-propanol / diethylamine in a volume ratio of 99:1:0.002 to 50:50:0.002 (v / v), or an aqueous methanol 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 reversed 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 resolution through a chiral chromatography column to obtain enantiomeric compounds.

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

[0034] In summary, the present invention provides an alkaloid compound isolated from Anacyclus pyrethrum roots, a preparation method and uses thereof. Using Anacyclus pyrethrum (L.) DC. roots as raw materials, solvent extraction, acid-base treatment, solvent extraction are carried out, and separation is carried out by two, three or four methods among silica gel column chromatography, preparative thin layer chromatography, Sephadex LH-20 column chromatography or high performance liquid chromatography (pHPLC). Thin layer chromatography or high performance liquid chromatography (HPLC) is used for detection and analysis to obtain 4 new skeleton alkaloid compounds. The in vitro anti-inflammatory activity of the compounds was determined, and the experimental results showed that the new skeleton alkaloid compounds isolated from Anacyclus pyrethrum roots have certain anti-inflammatory activities and can be used for the preparation of 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 of Compound 1 1 1H NMR spectrum;

[0039] Figure 5 is of Compound 1 13 13C NMR spectrum;

[0040] Figure 6 is of Compound 2 1 1H NMR spectrum;

[0041] Figure 7 is of Compound 2 13 13C NMR spectrum;

[0042] Figure 8 is the 1 1H NMR spectrum of Compound 3;

[0043] Figure 9 is the 13 13C NMR spectrum of Compound 3;

[0044] Figure 10 is the 1 1H NMR spectrum of Compound 4;

[0045] Figure 11 is the 13 13C NMR spectrum of Compound 4. Detailed implementation mode

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

[0047] According to the present invention, in the first aspect, a compound with anti-inflammatory effects is provided, and the structural formulas of the compounds (compounds of Formula 1, Formula 2, Formula 3 or Formula 4 respectively) are shown in the following figure;

[0048] .

[0049] Formula 1

[0050] .

[0051] Formula 2

[0052] .

[0053] Formula 3

[0054] .

[0055] Formula 4

[0056] Wherein:

[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-methano-cyclopenta[7,8]azabenz[4,5,6-ij]isoquinolin-2-yl)-2-methylpropyl-2-yl)acetamide (compound of Formula 1);

[0058] The compound (–)-1 is: N-(1-((8S,9aR,10aR)-2,2,5,5,8,12,12-heptamethyl-3,7-dioxo-1,2,3,4,5,6,7,8,9,11,12-decahydro-6H,13H-9a,14a-methano-cyclopenta[7,8]azoni[4,5,6-ij]isoquinolin-2-yl)-2-methylpropyl-2-yl)acetamide (the compound of formula 1);

[0059] The 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]indazol-10(1H)-one (the compound of formula 2);

[0060] The 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]indazol-10(1H)-one (the compound of formula 2);

[0061] The 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 (the compound of formula 3);

[0062] The 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 (the compound of formula 3);

[0063] The 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 (the compound of formula 4);

[0064] The 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 (the compound of formula 4).

[0065] The method for extraction and separation of the above alkaloid compounds is carried out according to the following steps:

[0066] Dry and crush Anake root, and use an ethanol aqueous solution with a volume fraction of 50 - 95% (v / v), methanol or chloroform as the solvent (the ratio of the weight of the medicinal material (Kg) to the solvent (L) is 1:1.5 - 1:4). Extract by cold soaking, percolation, heating under reflux or ultrasonic extraction, and concentrate under reduced pressure to recover the solvent to obtain an extract.

[0067] b. After suspending the total extract in step a in water, disperse and treat it with an acid such as hydrochloric acid with a concentration of 1 - 5% or sulfuric acid with a concentration of 1 - 5%. For the obtained acid aqueous layer, extract with dichloromethane to remove non - alkaloids, then adjust the pH to 10 - 12 with an alkali such as NaHCO3, Na2CO3, ammonia water or NaOH, and then extract with an organic solvent such as dichloromethane, ethyl acetate or n - butanol. Concentrate under reduced pressure to recover the organic solvent to obtain total alkaloids.

[0068] c. Separate the total alkaloids in step b by two, three or four methods among silica gel column chromatography, thin - layer chromatography, reversed - phase MCI column chromatography, Sephadex LH - 20 column chromatography and high - performance liquid chromatography.

[0069] Among them, for two separation methods:

[0070] The eluent for normal - phase silica gel column chromatography used is 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 for gradient elution, and then use reversed - phase silica gel or MCI column chromatography or semi - preparative high - performance liquid chromatography to obtain the compounds of formula 1, formula 2, formula 3 or formula 4.

[0071] For three separation methods:

[0072] The eluent for normal - phase silica gel column chromatography used is petroleum ether - ethyl acetate, dichloromethane - methanol or chloroform - methanol with a volume ratio of 100:1−0:1 for gradient elution. After that, through reversed - phase silica gel or MCI column chromatography, gradient elution is carried out with a methanol aqueous solution with a volume ratio (v / v) or an acetonitrile aqueous solution with a volume fraction of 20 - 100% (v / v). Then, use semi - preparative high - performance liquid chromatography, and use 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) as the eluent to obtain the compounds of formula 1, formula 2, formula 3 or formula 4.

[0073] For four separation methods:

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

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

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

[0077] The preparation method of the alkaloid compounds in Anaqigen described in step c is characterized in that the eluent for the high performance liquid chromatography method is an aqueous methanol 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-propanol with a volume ratio of 99 - 50% (v / v) or n-hexane / 2-propanol / diethylamine with a volume ratio of 99:1:0.002 - 50:50:0.002 (v / v / v) is used as the eluent, and isocratic or gradient elution is adopted.

[0078] According to another aspect of the present invention, there is provided a method for extracting and separating a compound with anti-inflammatory effect. The Anaqigen roots are dried and crushed, and an aqueous ethanol solution with a volume ratio of 50 - 95%, methanol or chloroform is used as the solvent (the ratio of the weight of the medicinal material (Kg) to the solvent (L) is 1:1.5 - 1:4). Cold maceration, percolation extraction, heating under reflux or ultrasonic extraction is adopted, and the solvent is recovered by decompression concentration to obtain an extract. After the total extract is suspended in water, it is dispersed and treated with an acid such as hydrochloric acid with a concentration of 1 - 5% or sulfuric acid with a concentration of 1 - 5%. The obtained acid aqueous layer is extracted with dichloromethane to remove non-alkaloids, and then the pH is adjusted to 10 - 12 with NaHCO3, Na2CO3, ammonia water or NaOH, and then extracted with an organic solvent such as chloroform, ethyl acetate or n-butanol. The organic solvent is recovered by decompression concentration to obtain total alkaloids. The obtained total alkaloids are separated by two, three or four methods among silica gel column chromatography, thin layer chromatography, Sephadex LH-20 column chromatography or high performance liquid chromatography. Detection and analysis are carried out by thin layer chromatography or high performance liquid chromatography analysis to obtain 8 new skeleton alkaloid compounds. The silica gel column chromatography used is normal pressure or pressurized column chromatography, and the filler used is normal phase silica gel or reverse phase silica gel, and dichloromethane and methanol with a volume ratio of 500:1 - 3:1; petroleum ether and ethyl acetate with a volume ratio of 1:0 - 3:1; or methanol water with 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 used is normal pressure chromatography, and the developing system is a mixture of dichloromethane and methanol, or a mixture of petroleum ether and acetone. The eluent of the Sephadex LH-20 column chromatography 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 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 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 the use of the compound with anti-inflammatory effect for preparing anti-inflammatory drugs.

[0080] For the new skeleton alkaloid compounds of the present invention, through comprehensive analysis by combining various spectroscopic analysis methods (high resolution mass spectrometry, ultraviolet spectroscopy, infrared spectroscopy, and nuclear magnetic resonance spectroscopy) and methods such as X-ray single crystal diffraction, the structures of Compounds 1 to 4 prepared in the examples are determined. The relative configurations of Compound 1, Compound 3 and Compound 4 are determined by X-ray single crystal diffraction, as Figure 1 , Figure 2 and Figure 3 shown.

[0081] Compound 1 (Anacyphrethine C): Yellow massive crystals; optical rotation value [α]25 D 128 (c 0.092, methanol, enantiomeric compound (+)-1); [α]25 D -128 (c 0.092, methanol, enantiomeric compound (-)-1); ultraviolet (methanol) λ max (log ε) 288 (3.92) nm, 417 (3.74) nm; infrared (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), enantiomeric 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, enantiomeric compound (-)-1; high resolution mass spectrometry m / z 496.3532 [M + H] + (calculated value C 30 H 45 O3N3 + , 496.3534). Its 1 H and 13 C NMR spectral data are shown in Table 1.

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

[0083]

[0084] Compound 2 (Anacyphrethine D): Yellow powder; optical rotation value [α]25 D 92 (c 0.08, methanol, enantiomeric compound (+)-2); [α]25 D -92 (c 0.066, methanol, enantiomeric compound (-)-2); ultraviolet (methanol) λ max(log ε) 268 (408) nm, 453 (4.14) nm; infrared (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), enantiomeric compound (-)-2; high resolution mass spectrometry m / z 438.3114 [M + H] + (calcd C 27 H 40 O2N3 + , 438.3115). Its 1 H and 13 C NMR spectral data are shown in Table 2.

[0085] Table 2 1 H and 13 C NMR data (CDCl3, 600 MHz)

[0086]

[0087] Compound 3 (Anacyphrethine E): colorless block crystals; optical rotation value [α]25 D -34 (c 0.2, methanol, enantiomeric compound (-)-3); [α]25 D 34 (c 0.2, methanol, enantiomeric compound (+)-3); ultraviolet (methanol) λ max (log ε) 260 (4.22) nm; infrared (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, enantiomeric 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 spectrometry m / z 331.2376 [M + H] + (calculated for C 20 H 31 O2N2 + , 331.2380). Its 1 H and 13 C NMR spectral 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, enantiomeric compound (+)-4); [α]25 D -66 (c 0.2, methanol, enantiomeric compound (-)-4); ultraviolet (methanol) λ max (log ε) 253 (3.86) nm; infrared (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, enantiomeric 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 spectrometry m / z 303.2063 [M + H] + (calculated for C 18 H 27 O2N2 + , 303.2067). Its 1 H and 13 C NMR spectral data are shown in Table 3.

[0089] Table 3 1 H and13 13C NMR data (CDCl3, 600 MHz)

[0090]

[0091] Generally speaking, compared with the prior art, the above technical solutions conceived by the present invention mainly have the following technical advantages:

[0092] (1) Compounds 1 to 4 provided in the present invention are compounds with a new skeleton. Compound 1 is a pair of highly conjugated diamino 6 / 6 / 6 / 6 / 5 pentacyclic alkaloid new skeleton enantiomers, with a unique 6,13-diazapentacyclo[10.3.3 1,3 .1.0 2, 7 .0 8,12 nonadecane ring system skeleton structure, having 3 discontinuous chiral stereocenters. Compound 2 is a pair of triamino 6 / 5 / 6 / 5 / 5 pentacyclic alkaloid new skeleton enantiomers, with a unique 4,6,17-triazapentacyclo[10.7.0.0 4,11 .0 5,9 .0 13,18 nonadecane ring system skeleton structure, having 3 discontinuous chiral stereocenters. Compound 3 is a pair of triamino 6 / 6 / 5 tricyclic alkaloid new skeleton enantiomers, with a unique 5,12-diazatricyclo[7.4.0 2,6 nonadecane ring system skeleton structure, having 3 discontinuous chiral stereocenters. Compound 4 is a pair of new pyridone-pyrrole skeleton enantiomers, having 1 chiral stereocenter.

[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 will be further described in detail below with reference to the drawings and embodiments, but this does not limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps, conditions, etc. of the present invention shall fall within the scope of the present invention.

[0095] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used 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] The dried Anacyclus pyrethrum (L.) DC. roots (15.0 kg) were pulverized, ultrasonically extracted with chloroform (30 L), and the extraction solution was concentrated under reduced pressure and combined to obtain the 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 acidic aqueous solution was adjusted to pH 10 with saturated NaHCO3 aqueous solution under ice-water bath and continuous stirring to obtain the alkalized solution. The alkalized solution was fully extracted with dichloromethane, and the dichloromethane extraction solutions were combined and dried to obtain the total alkaloids.

[0099] Example 2

[0100] The dried Anacyclus pyrethrum (L.) DC. roots (15.0 kg) were pulverized, extracted by percolation with 95% ethanol, and the extraction solution was concentrated under reduced pressure and combined to obtain the 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 acidic aqueous solution was adjusted to pH 10 with ammonia aqueous solution under ice-water bath and continuous stirring to obtain the alkalized solution. The alkalized solution was fully extracted with ethyl acetate, and the ethyl acetate extraction solutions were combined and dried to obtain the total alkaloids.

[0101] Example 3

[0102] The dried Anacyclus pyrethrum (L.) DC. roots (15.0 kg) were pulverized, extracted by percolation with 50% ethanol (40 L), and the extraction solution was concentrated under reduced pressure and combined to obtain the 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 acidic aqueous solution was adjusted to pH 10 with Na2CO3 aqueous solution under ice-water bath and continuous stirring to obtain the alkalized solution. The alkalized solution was fully extracted with n-butanol, and the n-butanol extraction solutions were combined and dried to obtain the total alkaloids.

[0103] Example 4

[0104] The dried Anacyclus pyrethrum (L.) DC. roots (15.0 kg) were pulverized, reflux-extracted with 75% ethanol (45 L), and the extraction solution was concentrated under reduced pressure and combined to obtain the 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 acidic aqueous solution was adjusted to pH 12 with NaOH aqueous solution under ice-water bath and continuous stirring to obtain the alkalized solution. The alkalized solution was fully extracted with dichloromethane, and the dichloromethane extraction solutions were combined and dried to obtain the total alkaloids.

[0105] Example 5

[0106] The dried Anacyclus pyrethrum (L.) DC. roots (15.0 kg) were crushed, extracted by cold maceration with methanol at room temperature, and the extraction solution was concentrated under reduced pressure. The combined extracts were obtained as 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 acidic aqueous solution was adjusted to pH 10 with saturated NaHCO3 aqueous solution under ice bath and continuous stirring conditions to obtain an alkalized solution. The alkalized solution was fully extracted with dichloromethane, and the combined dichloromethane extracts were dried to obtain the total alkaloids.

[0107] Example 6

[0108] The dried Anacyclus pyrethrum (L.) DC. roots (15.0 kg) were crushed, extracted by cold maceration with 22.5 L of methanol at room temperature, and the extraction solution was concentrated under reduced pressure. The combined extracts were obtained as 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 acidic aqueous solution was adjusted to pH 10 with saturated NaHCO3 aqueous solution under ice bath and continuous stirring conditions to obtain an alkalized solution. The alkalized solution was fully extracted with dichloromethane, and the combined dichloromethane extracts were dried to obtain the total alkaloids.

[0109] Example 7

[0110] The dried Anacyclus pyrethrum (L.) DC. roots (15.0 kg) were crushed, extracted by cold maceration with 22.5 L of methanol at room temperature, and the extraction solution was concentrated under reduced pressure. The combined extracts were obtained as 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 acidic aqueous solution was adjusted to pH 10 with saturated NaHCO3 aqueous solution under ice bath and continuous stirring conditions to obtain an alkalized solution. The alkalized solution was fully extracted with dichloromethane, and the combined dichloromethane extracts were dried to obtain the total alkaloids.

[0111] Example 8

[0112] The dried Anacyclus pyrethrum (L.) DC. roots (15.0 kg) were crushed, extracted by cold maceration with 22.5 L of methanol at room temperature, and the extraction solution was concentrated under reduced pressure. The combined extracts were obtained as 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 acidic aqueous solution was adjusted to pH 10 with saturated NaHCO3 aqueous solution under ice bath and continuous stirring conditions to obtain an alkalized solution. The alkalized solution was fully extracted with dichloromethane, and the combined dichloromethane extracts were dried to obtain the total alkaloids.

[0113] Example 9

[0114] Any of the total alkaloids in Examples 1-8 was mixed with silica gel of 100-200 mesh, and subjected to silica gel column chromatography, and then eluted with a gradient of dichloromethane / methanol (100:0~3:1, V / V). The same fractions were combined to obtain 6 fractions with increasing polarity, namely Fr.A, Fr.B, Fr.C, Fr.D, Fr.E and Fr.F; among them, the first fraction Fr.A (159.0 g) was mixed with silica gel of 100-200 mesh, and subjected to silica gel column chromatography, and then eluted with a gradient of petroleum ether / ethyl acetate (100:0~3:1, V / V). The same fractions were combined to obtain 6 fractions with increasing polarity, namely Fr.A1-Fr.A6; among them, the second fraction Fr.A2 (76.1 g) was subjected to reverse-phase MCI column chromatography and eluted with a gradient of acetonitrile / water (20:80~100:0, V / V). The same fractions were combined to obtain 7 sub-fractions with decreasing polarity, namely Fr.A21-Fr.A27; among them, the fraction Fr.A25 (10.0 g) was mixed with silica gel of 200-300 mesh, and subjected to silica gel column chromatography, and then eluted with a gradient of petroleum ether / ethyl acetate (10:1~3:1, V / V). The same fractions were combined to obtain 3 fractions with increasing polarity, namely Fr.A251-Fr.A253; the fraction Fr.A253 (9.0 g) was mixed with silica gel of 200-300 mesh, and subjected to silica gel column chromatography, and then eluted with a gradient of petroleum ether / ethyl acetate (10:1~3:1, V / V). The same fractions were combined to obtain 6 fractions with increasing polarity, namely Fr.A2531-Fr.A2533; among them, the fractions Fr.A251 and Fr.A2531 (136.3 mg) were combined and separated by semi-preparative high performance liquid chromatography on a C18 column. The mobile phase was methanol / water (10:100-100:0, V / V), and the flow rate was 3 mL / min to obtain Compound 4 (Anacyphrethines F, 45.0 mg, 0.000300%), and the retention time t R was 20.0 min; the racemate of Compound 4 was subjected to chiral resolution 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 the enantiomeric compound (+)-4 (13.0 mg, t R = 9.7 min) and the enantiomeric compound (-)-4 (13.3 mg, t R(= 11.2 min). Component Fr.A5 (22.9 g) was subjected to reversed-phase MCI column chromatography and eluted with a methanol / water gradient (10:90 - 100:0, V / V). The same components were combined to obtain nine sub-components Fr.A51−Fr.A59 with decreasing polarity; among them, component Fr.A58 (702.0 mg) was subjected to Sephadex LH-20 gel column chromatography and eluted with methanol to obtain four 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 with a mobile phase of methanol / water (70:30, V / V) and a flow rate of 3 mL / min to obtain compound 2 (Anacyphrethines D, 15.0 mg, 0.000300%), with a retention time t R of 51.9 min; the racemate of compound 2 was subjected to chiral resolution on a chiral column (DAICEL CORPORATION 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 the enantiomeric compound (+)-2 (6.5 mg, t R = 13.4 min) and the enantiomeric compound (-)-2 (6.3 mg, t R = 18.4 min).

[0115] Component Fr.B (179.1 g) was subjected to reversed-phase MCI column chromatography and eluted with a methanol / water gradient (20:80 - 100:0, V / V). The same components were combined to obtain two sub-components, Fr.B1 - Fr.B2, with decreasing polarity. Among them, component Fr.B1 (76.1 g) was subjected to silica gel column chromatography and then eluted with a petroleum ether / ethyl acetate gradient (1:0 - 3:1, V / V). The same components were combined to obtain four components, Fr.B1a - Fr.B1d, with increasing polarity. Among them, component Fr.B1b (18.6 g) was subjected to reversed-phase MCI column chromatography and eluted with a methanol / water gradient (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 g) was subjected to normal-phase silica gel column chromatography and eluted with dichloromethane / methanol (500:1 - 10:1, V / V) to obtain three sub-components, Fr.B1b41 - Fr.B1b44, with increasing polarity. Component Fr.B1b42 (2.0 g) was subjected to Sephadex LH-20 gel column chromatography and eluted with methanol to obtain two sub-components, Fr.B1b42a - Fr.B1b42b, with decreasing molecular weight. Component Fr.B1b42b (1.9 g) was subjected to reversed-phase C18 silica gel column chromatography and eluted with an acetonitrile / water gradient (20:80 - 100:0, V / V). The same components were combined to obtain eleven sub-components, Fr.B1b42b1 - Fr.B1b42b11, with decreasing polarity. Component Fr.B1b42b1 (552.0 mg) was subjected to normal-phase silica gel column chromatography and eluted with dichloromethane / methanol (500:1 - 5:1, V / V) to obtain five sub-components, Fr.B1b42b1a - Fr.B1b42b1e, with increasing polarity. Component Fr.B1b42b1c (183.2 mg) was subjected to Sephadex LH-20 gel column chromatography and eluted with methanol to obtain four sub-components, Fr.B1b42b1c1 - Fr.B1b42b1c4, with decreasing molecular weight. Component Fr.B1b42b1c2 (127.9 mg) was subjected to reversed-phase C 18 Silica gel column chromatography was carried out, eluting with a methanol / water gradient (10:90 - 100:0, V / V). The same components were combined to obtain four sub-components, Fr.B1b42b1c2a - Fr.B1b42b1c2d, with decreasing polarity. Component Fr.B1b42b1c2d (15.5 mg) was subjected to semi-preparative high-performance liquid chromatography C 18 Column separation was performed with a mobile phase of methanol / water (36:64, V / V) and a flow rate of 3 mL / min to obtain compound 3 (Anacyphrethines C, 17.7 mg, 0.000113%); retention time t Rwas 17.0 min; the racemate of compound 3 was subjected to chiral resolution 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 the enantiomeric compound (+)-3 (1.5 mg, t R = 17.6 min) and the enantiomeric compound (-)-3 (1.4 mg, t R = 12.6 min). The fraction Fr.B1b5 (2.6 g) was subjected to normal-phase silica gel column chromatography and eluted with dichloromethane / methanol (500:1 - 5:1, V / V) to obtain 3 sub-fractions Fr.B1b51−Fr.B1b53 with increasing polarity; among them, the fraction Fr.B1b52 (983.0 mg) was subjected to Sephadex LH-20 gel column chromatography and eluted with methanol to obtain 2 sub-fractions Fr.B1b52a−Fr.B1b52b with decreasing molecular weight; the fraction Fr.B1b52b (736.9 mg) was subjected to reverse-phase C18 silica gel column chromatography and eluted with a methanol / water gradient (20:80 - 100:0, V / V), and the same components were combined to obtain 6 sub-fractions Fr.B1b52b1−Fr.B1b52b6 with decreasing polarity; the fraction Fr.B1b52b5 (175.0 mg) was subjected to normal-phase silica gel column chromatography and eluted with dichloromethane / methanol (500:1 - 5:1, V / V) to obtain 3 sub-fractions Fr.B1b52b5a−Fr.B1b52b5c with increasing polarity; among them, the fraction Fr.B1b52b5b (170.0 mg) was subjected to Sephadex LH-20 gel column chromatography and eluted with methanol to obtain 3 sub-fractions Fr.B1b52b5b1−Fr.B1b52b5b3 with decreasing molecular weight; the fraction Fr.B1b52b5b2 (123.0 mg) was subjected to normal-phase silica gel column chromatography and eluted with dichloromethane / methanol (300:1 - 5:1, V / V) to obtain 2 sub-fractions Fr.B1b52b5b2a−Fr.B1b52b5b2b with increasing polarity; the fraction Fr. B1b52b5b2a (92.5 mg) was separated by semi-preparative high performance liquid chromatography C 18 column, the mobile phase was acetonitrile / water (20:100 - 100:0, V / V), the flow rate was 3 mL / min, and compound 1 (Anacyphrethines C, 17.7 mg, 0.000113%) was obtained; the retention time t Rwas 27.3 min; the racemate of Compound 1 was subjected to chiral resolution on a chiral column (DAICEL CORPORATION 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 the enantiomeric compound (+)-1 (6.2 mg, t R = 30.8 min) and the enantiomeric compound (-)-1 (6.0 mg, t R = 33.3 min).

[0116] Example 10

[0117] Any of the total alkaloids in the above-mentioned total alkaloids of Examples 1-8 was mixed with silica gel of 100-200 mesh, and subjected to silica gel column chromatography, and then gradient elution was carried out with dichloromethane / methanol (100:0~3:1, V / V). 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; among them, the first component Fr.A (159.0 g) was subjected to repeated silica gel column chromatography, and 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 was used as the eluent for gradient elution to obtain Compound 4 (Anacyphrethines F, 45.0 mg, 0.000300%) and Compound 2 (Anacyphrethines D, 15.0 mg, 0.000300%); the racemate of Compound 4 was subjected to chiral resolution on a chiral 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 enantiomeric compound (+)-4 (13.0 mg, t R = 9.7 min) and the enantiomeric compound (-)-4 (13.3 mg, t R = 11.2 min). The racemate of Compound 2 was subjected to chiral resolution on a chiral 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 enantiomeric compound (+)-2 (6.5 mg, t R= 13.4 min) and the enantiomeric 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, eluted with petroleum ether-ethyl acetate with a volume ratio of 100:0 - 3:1, dichloromethane-methanol with a ratio of 500:1 - 3:1 or petroleum ether / acetone with a volume ratio of 50:1 - 0:1 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 resolution on a chiral column (DAICEL CORPORATION Chiralpak ID 5 µm 10 × 250 mm; solvent: n-hexane / 2-propanol = 99:1 - 50:50; column temperature: 25 °C; flow rate: 3 ml / min; detection wavelength: 254 nm) to obtain the enantiomeric compound (+)-3 (1.5 mg, t R = 17.6 min) and the enantiomeric compound (-)-3 (1.4 mg, t R = 12.6 min); the racemate of compound 1 was subjected to chiral resolution on a chiral column (DAICEL CORPORATION Chiralpak ID 5 µm 10 × 250 mm; solvent: n-hexane / 2-propanol / diethylamine = 99:1:0.002 - 50:50:0.002; column temperature: 25 °C; flow rate: 3 ml / min; detection wavelength: 254 nm) to obtain the enantiomeric compound (+)-1 (6.2 mg, t R = 30.8 min) and the enantiomeric compound (-)-1 (6.0 mg, t R = 33.3 min).

[0119] Example 11

[0120] Anti-inflammatory activity of the novel skeleton alkaloid compounds isolated from Anacyclus roots according to the present invention

[0121] 1. Cell culture:

[0122] Mouse macrophages Raw264.7 (purchased from BeNa Culture Collection, BNCC) were cultured in high-glucose Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) (purchased from Gibco, USA), 1% penicillin and streptomycin, in an incubator at 37°C and 5% CO2;

[0123] 2. Test for the effect of the compound described in the present invention on cell viability:

[0124] The compound of the present invention was dissolved in dimethyl sulfoxide (DMSO). Raw264.7 cells in the logarithmic growth phase with good growth state were seeded into a 96-well plate at a density of 5 × 10 3 cells / well. Different concentrations of various compounds were added to the experimental groups, 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-disulfophenyl)-2H-tetrazolium monosodium salt (CCK-8 reagent) was added to each well. The absorbance at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) 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] Those skilled in the art can easily understand 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 principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Alkaloid compounds extracted from Anaqigen: 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.

2. The alkaloid compound according to claim 1, which is selected from compound (+)-3 or compound (–)-3, wherein: 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 (–)-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, which respectively have the following structural formulas: or 。 3. A method for extracting the alkaloid compound according to claim 1 or 2 from Anaqigen, which comprises the following steps: a. Dry and pulverize Anaqigen roots, use an ethanol - water solution with a volume ratio of 50 - 95%, methanol or chloroform as a solvent, and the ratio of Anaqigen roots in kg by weight to the solvent in L by volume is 1:1.5 - 1:

4. Use cold soaking, percolation extraction, heating under reflux or ultrasonic extraction, and concentrate under reduced pressure to recover the solvent to obtain an extract; b. After suspending the total extract in step a in water, perform acid dispersion treatment. For the obtained acid - water layer, extract with dichloromethane to remove non - alkaloids, then adjust the pH to 10 - 12 with alkali, and 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; and c. Subject the total alkaloids in step b to gradient or isocratic elution using a normal - phase silica gel column chromatography, and then separate 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. The eluent used in the normal - phase silica gel column chromatography is petroleum ether and ethyl acetate with a volume ratio of 100:0 to 3:1, dichloromethane and methanol with a volume ratio of 500:1 to 3:1, petroleum ether and acetone with a volume ratio of 50:1 to 0:1, or methanol and water with a volume ratio of 1:9 - 1:

0. The eluent used in the reverse - phase silica gel or reverse - phase MCI column chromatography is an aqueous methanol solution with a volume ratio of 10 - 100% or an aqueous acetonitrile 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 method 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 an aqueous methanol solution with a volume ratio of 20 - 100%.

4. A method for extracting the alkaloid compound according to claim 1 or 2 from Anaqigen, which comprises the following steps: a. Dry and pulverize Anaqigen, and use an ethanol aqueous solution with a volume ratio of 50 - 95%, methanol or chloroform as the solvent. The ratio of Anaqigen in kg by weight to the solvent in L by volume is 1:1.5 - 1:

4. Use cold maceration, percolation extraction, heating under reflux or ultrasonic extraction, and concentrate under reduced pressure to recover the solvent to obtain an extract. b. After suspending the total extract in step a in water, perform acid dispersion treatment. For the obtained acid aqueous layer, use dichloromethane to extract and remove non - alkaloids, then adjust the pH to 10 - 12 with alkali, and then extract with organic solvents such as dichloromethane, ethyl acetate or n - butanol. Concentrate under reduced pressure to recover the organic solvent to obtain total alkaloids; and c. Use normal - phase silica gel column chromatography for gradient or isocratic elution of the total alkaloids in step b, then use reverse - phase silica gel column chromatography or reverse - phase MCI column chromatography for gradient elution, and then use semi - preparative high - performance liquid chromatography for separation to obtain the alkaloid compounds. The eluent used in the normal - phase silica gel column chromatography is petroleum ether and ethyl acetate, dichloromethane and methanol or chloroform and methanol with a volume ratio of 100:1 to 0:

1. The eluent used in the reverse - phase silica gel or reverse - phase MCI column chromatography is an aqueous methanol solution with a volume ratio of 10 - 100% or an aqueous acetonitrile 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 an aqueous methanol solution with a volume ratio of 10 - 100%.

5. A method for extracting the alkaloid compounds according to claim 1 or 2 from Anaqigen, comprising the following steps: a. Dry and pulverize Anaqigen, and use an ethanol aqueous solution with a volume ratio of 50 - 95%, methanol or chloroform as the solvent. The ratio of Anaqigen in kg by weight to the solvent in L by volume is 1:1.5 - 1:

4. Use cold maceration, percolation extraction, heating under reflux or ultrasonic extraction, and concentrate under reduced pressure to recover the solvent to obtain an extract. b. After suspending the total extract in step a in water, perform acid dispersion treatment. For the obtained acid aqueous layer, use dichloromethane to extract and remove non - alkaloids, then adjust the pH to 10 - 12 with alkali, and then extract with organic solvents such as dichloromethane, ethyl acetate or n - butanol. Concentrate under reduced pressure to recover the organic solvent to obtain total alkaloids; and c. Subject the total alkaloids in step b to gradient or isocratic elution using a normal-phase silica gel column chromatography, then subject them to Sephadex LH-20 column chromatography, then perform gradient elution using a reverse-phase silica gel column chromatography or a reverse-phase MCI column chromatography, and finally separate them 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 with 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 with a volume ratio of 100:1 to 0:1, the Sephadex LH-20 column chromatography uses methanol gradient or isocratic elution, the eluent used in the reverse-phase silica gel or reverse-phase MCI column chromatography is an aqueous methanol solution with a volume ratio of 10 - 100% or an aqueous acetonitrile 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-propanol with a volume ratio of 99 - 50%, n-hexane / 2-propanol / diethylamine with a volume ratio of 99:1:0.002 to 50:50:0.002, or an aqueous methanol 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 method according to any one of claims 3 to 5, wherein in step b, the base used is NaHCO3, Na2CO3, ammonia water, or NaOH.

8. The method according to any one of claims 3 to 5, wherein in step c, the silica gel column chromatography is atmospheric pressure or pressurized column chromatography, and the packing 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 a step of subjecting the compound obtained as a racemate to chiral resolution using a chiral chromatography column to obtain an enantiomeric compound.

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

Images