CN114805465B - Triterpene compound, preparation method and application thereof - Google Patents
Triterpene compound, preparation method and application thereof Download PDFInfo
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- CN114805465B CN114805465B CN202110060416.2A CN202110060416A CN114805465B CN 114805465 B CN114805465 B CN 114805465B CN 202110060416 A CN202110060416 A CN 202110060416A CN 114805465 B CN114805465 B CN 114805465B
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- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- -1 Triterpene compound Chemical class 0.000 title abstract description 8
- 150000001875 compounds Chemical class 0.000 claims abstract description 53
- 239000003814 drug Substances 0.000 claims abstract description 14
- 235000003332 Ilex aquifolium Nutrition 0.000 claims abstract description 12
- 235000002296 Ilex sandwicensis Nutrition 0.000 claims abstract description 12
- 235000002294 Ilex volkensiana Nutrition 0.000 claims abstract description 12
- 229940079593 drug Drugs 0.000 claims abstract description 11
- 150000003839 salts Chemical class 0.000 claims abstract description 11
- 206010061218 Inflammation Diseases 0.000 claims abstract description 9
- 230000004054 inflammatory process Effects 0.000 claims abstract description 9
- 239000000243 solution Substances 0.000 claims description 34
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 33
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 28
- 239000002158 endotoxin Substances 0.000 claims description 23
- 229920006008 lipopolysaccharide Polymers 0.000 claims description 23
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Substances OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 17
- 238000004440 column chromatography Methods 0.000 claims description 13
- GLYLMXARZJNUEY-UHFFFAOYSA-N dichloromethane;methanol;hydrate Chemical compound O.OC.ClCCl GLYLMXARZJNUEY-UHFFFAOYSA-N 0.000 claims description 13
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 12
- 238000010828 elution Methods 0.000 claims description 12
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- 239000008194 pharmaceutical composition Substances 0.000 claims description 7
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- HTSGKJQDMSTCGS-UHFFFAOYSA-N 1,4-bis(4-chlorophenyl)-2-(4-methylphenyl)sulfonylbutane-1,4-dione Chemical compound C1=CC(C)=CC=C1S(=O)(=O)C(C(=O)C=1C=CC(Cl)=CC=1)CC(=O)C1=CC=C(Cl)C=C1 HTSGKJQDMSTCGS-UHFFFAOYSA-N 0.000 claims description 6
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- 238000000034 method Methods 0.000 claims description 5
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- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 description 34
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- MSQDVGOEBXMPRF-UHFFFAOYSA-N cyclohexane;propan-2-one Chemical compound CC(C)=O.C1CCCCC1 MSQDVGOEBXMPRF-UHFFFAOYSA-N 0.000 description 12
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- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 6
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- NLFBCYMMUAKCPC-KQQUZDAGSA-N ethyl (e)-3-[3-amino-2-cyano-1-[(e)-3-ethoxy-3-oxoprop-1-enyl]sulfanyl-3-oxoprop-1-enyl]sulfanylprop-2-enoate Chemical compound CCOC(=O)\C=C\SC(=C(C#N)C(N)=O)S\C=C\C(=O)OCC NLFBCYMMUAKCPC-KQQUZDAGSA-N 0.000 description 3
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- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- FKLJPTJMIBLJAV-UHFFFAOYSA-N Compound IV Chemical compound O1N=C(C)C=C1CCCCCCCOC1=CC=C(C=2OCCN=2)C=C1 FKLJPTJMIBLJAV-UHFFFAOYSA-N 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
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- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 2
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- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 description 2
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- JUJWROOIHBZHMG-QYKNYGDISA-N 2-deuteriopyridine Chemical compound [2H]C1=CC=CC=N1 JUJWROOIHBZHMG-QYKNYGDISA-N 0.000 description 1
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- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
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- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
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- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
- C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
- C07J71/0005—Oxygen-containing hetero ring
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Rheumatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Pain & Pain Management (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the technical field of medicines, relates to a triterpene compound, a preparation method and application thereof, and in particular relates to a triterpene compound obtained from roughhaired holly, a preparation method thereof and application thereof in preparing anti-inflammatory medicines. The invention provides a compound shown in the formula I-V or pharmaceutically acceptable salt thereof, the structure of the compound is as follows, and the invention also provides application of the compound shown in the formula I-V or pharmaceutically acceptable salt thereof or medicine and composition thereof in preparing medicines for preventing and/or treating inflammation. The inflammation can be gastritis, pneumonia, hepatitis, mastitis or trauma inflammation, etc.
Description
Technical Field
The invention belongs to the technical field of medicines, relates to a triterpene compound, a preparation method and application thereof, and in particular relates to a triterpene compound obtained from roughhaired holly, a preparation method thereof and application thereof in preparing anti-inflammatory medicines.
Background
The holly root and rhizome are dry roots and rhizomes of holly leaf Ilex (Ilex asprilla (hook. Et Arn.) champ. Ex benth.) which are the plants of the family Ilex, and are called star tree, licorice, arisaema with aliquoted names, and are the traditional Chinese medicines in the south of the Ling of China, which are originally carried in the "Shengcao Yam" preparation. The roughhaired holly root has bitter taste and cold property, has the effects of clearing heat and detoxicating, promoting the production of body fluid and relieving sore throat, and removing blood stasis and relieving pain, is generally used together with other traditional Chinese medicines, and is a main prescription component of various commercially available Chinese patent medicines for relieving fever and pain, removing dampness and removing stagnation, such as Ganmaoling granules, exogenous granules, green plum cold granules, guangdong herbal tea granules, shaxi herbal tea and the like.
For decades, students at home and abroad have studied the chemical components of roughhaired holly, and triterpenes and glycosides thereof are the most main components in roughhaired holly, and structural types such as steroids, glycosides, lignans, flavonoids and the like. Modern pharmacological researches have shown that triterpenes in roughhaired holly have the effects of anti-inflammatory, lipid-lowering, antibacterial and antitumor. However, little research has been done on the basis of the anti-inflammatory pharmacodynamic substances of holly.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention takes Ilex asprilla (hook. Et Arn.) champ. Ex benth.) as a research object, separates and purifies ethanol extract thereof to obtain five novel triterpene compounds, and carries out structure identification and biological activity research on the compounds, so that the compounds have good anti-inflammatory activity and potential medicinal value.
The invention is realized by the following technical scheme:
the invention provides compounds represented by formulas I-V or pharmaceutically acceptable salts thereof;
the compounds of the formulas I-V are named ileospside A, 4-methyl-11 alpha, 12 alpha-epoxy-2-hydroxy-3, 5-dioxa-1, 4-dine-28-oic acid gamma-lactone, 4-methyl-11 alpha, 12 alpha-epoxy-2-hydroxy-3, 5-dioxa-1,4,20, (29) -trie-28-oic acid gamma-lactone, 11 alpha, 12 alpha-epoxy-3 beta-hydroxy-24-norula-4 (23) -en-28,13 beta-olide respectively.
The invention also provides a preparation method of the compound of the formula I-V or pharmaceutically acceptable salt thereof, which comprises the following steps:
(1) Extracting dry root of roughhaired holly with ethanol, concentrating to obtain total extract;
(2) Suspending the total extract with water, loading onto macroporous resin adsorption column, and gradient eluting with 0-95% ethanol to obtain five fractions of IA-A, IA-B, IA-C, IA-D, IA-E;
(3) Separating IA-C and IA-E by silica gel column chromatography, and gradient eluting with dichloromethane/chloroform-methanol system (100:0-0:100) to obtain fractions IA-C1-IA-C12 and IA-EA-IA-EN;
(4) Separating IA-C9 by ODS column chromatography, and gradient eluting with methanol-water system (10% -100%) to obtain IA-C1-A-I;
(5) Separating IA-C9-D and IA-C9-E by silica gel column chromatography, and gradient eluting with dichloromethane-methanol-water system (9:1:0.1-6:4:0.8) to obtain IA-C9-D1-IA-C9-D4 and IA-C9-E1;
(6) Crystallizing IA-C9-D4 in methanol to obtain a compound II; further separating IA-C9-E3 by High Performance Liquid Chromatography (HPLC) to obtain compound I;
(7) Further separating and purifying by sephadex LH-20 column chromatography by IA-EC, and performing isocratic elution by a methylene dichloride-methanol system (volume ratio is 1:1) to obtain fraction IA-EC-2;
(8) Separating IA-EC-2 by silica gel column chromatography, and sequentially performing gradient elution by using a cyclohexane-acetone system (volume ratio is 100:0-0:100) to obtain IA-EC-21-IA-EC-29;
(9) Separating IA-EC-26 by ODS column chromatography, and sequentially performing gradient elution by using a methanol-water system (the volume percentage content is 50% -100%), so as to obtain IA-EC-261-IA-EC-265;
(10) Further separation of IA-EC-263 to produce a high performance liquid phase (HPLC) to give compound III;
(11) Separating IA-EC-27 by ODS column chromatography, and sequentially performing gradient elution by using a methanol-water system (the volume percentage content is 50% -100%) to obtain IA-EC-271-IA-EC-275;
(12) IA-EC-274 is subjected to separation for preparing a High Performance Liquid (HPLC) to obtain a compound IV and a compound V.
Wherein, in the step (1), the volume concentration of the ethanol is 60-90%, and the ethanol is extracted for 1-3 times by adopting heating reflux for 1-3 hours each time;
the mobile phase in the step (6) is an acetonitrile-water solution with the concentration of 10-20%;
the mobile phase in step (10) is 60-70% methanol-water solution;
the mobile phase in step (12) is 60-75% methanol-water solution.
The invention provides a pharmaceutical composition, which comprises one or more of compounds shown in the formulas I-V or pharmaceutically acceptable salts thereof.
The invention provides a pharmaceutical composition, which comprises one or more of the compounds shown in the formulas I-V or pharmaceutically acceptable salts thereof or pharmaceutically acceptable carriers or excipients.
The invention also provides application of the compound shown in the formulas I-V or pharmaceutically acceptable salts thereof or medicines and compositions thereof in preparing medicines for preventing and/or treating inflammation. The inflammation can be gastritis, pneumonia, hepatitis, mastitis or trauma inflammation, etc.
Description of the drawings:
FIG. 1 shows the effect of compounds I and II on LPS-induced expression of iNOS proteins and COX-2 proteins;
wherein LPS refers to lipopolysaccharide; iNOS refers to inducible nitric oxide synthase; COX-2 refers to cyclooxygenase 2.
FIG. 2 shows the effect of compounds I and II on LPS-induced degradation of IκB- α protein;
wherein IκB- α refers to human nuclear factor κB inhibitor protein α.
FIG. 3 shows the effect of compounds I and II on LPS-induced phosphorylation of ERK1/2, JNK, p38 proteins.
The specific embodiment is as follows:
wherein ERK1/2 refers to extracellular regulated protein kinase; JNK refers to stress activated protein kinase; p38 refers to mitogen-activated protein kinase.
Example 1 preparation of ethanol extract of Ilicis Asprellae
18.9kg of dry root of roughhaired holly is heated and extracted for 2 times by 10 times of 60% ethanol under reflux for 2 hours each time, the extracting solutions are combined, the solvent is recovered under reduced pressure, and the total extract is obtained after concentration, namely 2.56kg. Taking 1.2kg of extract, suspending with 5 times of water (6L), separating by D101 macroporous adsorption resin column chromatography, and eluting with ethanol-water gradient to obtain five fractions of water eluting part (IA-A, 508.0 g), 30% ethanol eluting part (IA-B, 73.0 g), 50% ethanol eluting part (IA-C, 123.7 g), 70% ethanol eluting part (IA-D, 260.6 g) and 95% ethanol eluting part (IA-E, 85.1 g).
Method for preparing the Compound of example 2
The 50% ethanol elution fraction (IA-C) of the ethanol extract of Ilicis Asprellae was separated by column chromatography on silica gel, and gradient elution was performed sequentially with a methylene chloride-methanol system (volume ratio of methylene chloride to methanol: 100:0, 99:1, 98:2, 97:3, 95:5, 93:7,9:1,8:2,7:3,6:4,1:1, 0:100), each system was washed with 4 retention volumes, each volume was 1000mL. According to the thin layer chromatography behavior analysis, fraction IA-C1 is obtained in methylene dichloride-methanol (volume ratio of 100:0 and 99:1); obtaining a fraction IA-C2 in methylene dichloride-methanol (volume ratio of 99:1); obtaining a fraction IA-C3 in methylene dichloride-methanol (volume ratio 98:2); obtaining fraction IA-C4 in methylene dichloride-methanol (volume ratio 97:3); obtaining fraction IA-C5 in methylene dichloride-methanol (volume ratio 95:5); obtaining fraction IA-C6 in methylene dichloride-methanol (volume ratio 93:7); obtaining a fraction IA-C7 in methylene dichloride-methanol (volume ratio 9:1); obtaining a fraction IA-C8 in methylene dichloride-methanol (volume ratio of 8:2); obtaining fraction IA-C9 in methylene dichloride-methanol (volume ratio 7:3); obtaining a fraction IA-C10 in methylene dichloride-methanol (volume ratio of 6:4); obtaining a fraction IA-C11 in methylene dichloride-methanol (volume ratio of 1:1); fractions IA-C12 were obtained in methylene chloride-methanol (volume ratio 0:100). A total of 12 fractions were obtained, and each fraction was freeze-dried.
IA-C9 (38.0 g) was subjected to ODS column chromatography and eluted with a gradient of methanol-water systems (10%, 20%,30%,40%,50%,60%,70%,80%,100% by volume of methanol-water) in this order, 4 retention volumes were washed with 300mL of each system. According to the analysis of the color development behavior of the thin layer chromatography, obtaining a fraction IA-C9-A in a methanol-water solution with the volume percentage content of 10%; obtaining a fraction IA-C9-B in a methanol-water solution with the volume percentage content of 20%; obtaining a fraction IA-C9-C in a 30% by volume methanol-water solution; obtaining a fraction IA-C9-D in a 40% by volume methanol-water solution; obtaining a fraction IA-C9-E in a methanol-water solution with the volume percentage content of 50%; obtaining a fraction IA-C9-F in a 60% by volume methanol-water solution; obtaining a fraction IA-C9-G in a methanol-water solution with the volume percentage content of 70%; obtaining a fraction IA-C9-H in a methanol-water solution with the volume percentage content of 80%; the fraction IA-C9-I is obtained in a methanol-water solution with a volume percentage of 100%. A total of 9 fractions were obtained, and each fraction was freeze-dried.
IA-C9-D (825.9 mg) was separated by silica gel column chromatography and eluted sequentially with a gradient of methylene chloride-methanol-water system (methylene chloride-methanol-water volume ratio 9:1:0.1,8:2:0.2,7:3:0.5, 6:4:0.8), each system washed with 4 retention volumes of 1000mL each. According to the thin layer chromatography behavior analysis, obtaining fraction IA-C9-D1 in methylene dichloride-methanol-water (volume ratio 9:1:0.1); obtaining a fraction IA-C9-D2 in methylene dichloride-methanol (volume ratio of 8:2:0.2); obtaining a fraction IA-C9-D3 in methylene dichloride-methanol (volume ratio of 7:3:0.5); fractions IA-C9-D4 were obtained in methylene chloride-methanol (6:4:0.8 by volume). A total of 4 fractions were obtained, and each fraction was freeze-dried.
IA-C9-D4 (281.5 mg) was crystallized from methanol as compound II (178.3 mg).
IA-C9-E (4.7 g) was separated by silica gel column chromatography and eluted sequentially with a gradient of methylene chloride-methanol-water system (methylene chloride-methanol-water volume ratio 9:1:0.1,8:2:0.2,7:3:0.5, 6:4:0.8), 4 retention volumes per system, 1000mL each. According to the thin layer chromatography behavior analysis, obtaining fraction IA-C9-E1 in methylene dichloride-methanol-water (volume ratio 9:1:0.1); obtaining a fraction IA-C9-E2 in methylene dichloride-methanol (volume ratio of 8:2:0.2); obtaining a fraction IA-C9-E3 in methylene dichloride-methanol (volume ratio of 7:3:0.5); fractions IA-C9-E4 were obtained in methylene chloride-methanol (6:4:0.8 by volume). A total of 4 fractions were obtained, and each fraction was freeze-dried.
A further separation of the preparative High Performance Liquid (HPLC) was carried out on IA-C9-E3 (2.8 g), the HPLC preparation was carried out with a differential detector as the detection means and with an acetonitrile-water solution of 19% by volume as the eluent, the flow rate was 4mL/min, and the chromatographic peak was collected for 28min to give Compound I (226.6 mg).
The 95% ethanol elution fraction (IA-E) of the ethanol extract of Ilicis Asprellae was separated by column chromatography on silica gel, and gradient elution was performed sequentially with a methylene chloride-methanol system (volume ratio of methylene chloride to methanol: 100:0, 99:1, 98:2, 97:3, 95:5, 93:7,9:1,8:2,7:3,6:4,1:1, 0:100), each system was washed with 4 retention volumes, each volume was 1000mL. Obtaining fractions IA-EA, IA-EB and IA-EC in methylene chloride-methanol (volume ratio 100:0) according to thin layer chromatography behavior analysis; obtaining fraction IA-ED in methylene dichloride-methanol (volume ratio of 99:1); obtaining fraction IA-EE in methylene dichloride-methanol (volume ratio 98:2); obtaining fraction IA-EF in methylene dichloride-methanol (volume ratio 97:3); obtaining fraction IA-EG in methylene dichloride-methanol (volume ratio of 95:5); obtaining a fraction IA-EH in methylene dichloride-methanol (volume ratio 93:7); obtaining a fraction IA-EI in methylene dichloride-methanol (volume ratio 9:1); obtaining a fraction IA-EJ in methylene dichloride-methanol (volume ratio of 8:2); obtaining a fraction IA-EK in methylene dichloride-methanol (volume ratio of 7:3); obtaining fraction IA-EL in methylene dichloride-methanol (volume ratio 6:4); obtaining fraction IA-EM in methylene dichloride-methanol (volume ratio 1:1); fractions IA-EN were obtained in methylene chloride-methanol (volume ratio 0:100). A total of 14 fractions were obtained, and each fraction was freeze-dried.
Further sephadex LH-20 column chromatography purification was performed on IA-EC (3.8 g), isocratic elution was performed on a methylene chloride-methanol system (volume ratio 1:1) to give fraction IA-EC-2, which was freeze-dried.
IA-EC-2 (2.9 g) was separated by column chromatography on silica gel, gradient eluted sequentially with cyclohexane-acetone systems (cyclohexane-acetone volume ratio 100:0, 99:1, 98:2, 97:3, 95:5, 93:7,9:1,8:2,7:3,6:4,1:1, 0:100), each system washed with 4 retention volumes of 1000mL each volume. Obtaining fraction IA-EC-21 in cyclohexane-acetone (volume ratio 100:0) according to thin layer chromatography behavior analysis; obtaining fraction IA-EC-22 in cyclohexane-acetone (volume ratio 99:1); obtaining fraction IA-EC-23 in cyclohexane-acetone (volume ratio 98:2 and 97:3); fraction IA-EC-24 was obtained in cyclohexane-acetone (volume ratios 95:5 and 93:7); obtaining fraction IA-EC-25 in cyclohexane-acetone (volume ratio 9:1); obtaining fraction IA-EC-26 in cyclohexane-acetone (volume ratio 8:2); obtaining fraction IA-EC-27 in cyclohexane-acetone (volume ratio 7:3); obtaining fraction IA-EC-28 in cyclohexane-acetone (volume ratio 6:4); fraction IA-EC-29 was obtained in cyclohexane-acetone (volume ratio 1:1). A total of 9 fractions were obtained, and each fraction was freeze-dried.
IA-EC-26 (773.2 mg) was separated by ODS column chromatography, and gradient elution was sequentially carried out with methanol-water systems (50%, 60%,70%,80%,100% by volume of methanol-water), each system was washed with 4 retention volumes, and each volume was 300mL. According to the analysis of the color development behavior of the thin layer chromatography, the fraction IA-EC-261 is obtained in a methanol-water solution with the volume percentage content of 50%; obtaining a fraction IA-EC-262 in a 60% by volume methanol-water solution; obtaining a fraction IA-EC-263 in a methanol-water solution with a volume percentage of 70%; obtaining a fraction IA-EC-264 in a methanol-water solution with the volume percentage content of 80%; fraction IA-EC-265 was obtained in a 100% by volume methanol-water solution. A total of 5 fractions were obtained, and each fraction was freeze-dried.
IA-EC-263 (82.7 mg) was further subjected to separation to prepare a High Performance Liquid (HPLC) by using a differential detector as a detection means, and by using 70% by volume of methanol-water solution as an eluent to prepare the compound III (32.2 mg) by HPLC at a flow rate of 4mL/min and collecting a chromatographic peak for 35 min.
IA-EC-27 (657.8 mg) was subjected to ODS column chromatography, and gradient elution was sequentially carried out with methanol-water systems (50%, 60%,70%,80%,100% by volume of methanol-water), each system was washed with 4 retention volumes, and each volume was 300mL. According to the analysis of the color development behavior of the thin layer chromatography, obtaining a fraction IA-EC-271 in a methanol-water solution with the volume percentage content of 50%; obtaining a fraction IA-EC-272 in a 60% by volume methanol-water solution; obtaining a fraction IA-EC-273 in a methanol-water solution with a volume percentage of 70%; obtaining a fraction IA-EC-274 in a methanol-water solution with a volume percentage of 80%; fraction IA-EC-275 was obtained in a 100% by volume methanol-water solution. A total of 5 fractions were obtained, and each fraction was freeze-dried.
IA-EC-274 (128.1 mg) was further subjected to separation of preparative High Performance Liquid (HPLC) using a differential detector as detection means and methanol-water solution with a volume percent of 75% as eluent at a flow rate of 4mL/min to collect chromatographic peaks of 14 and 32min to give compound IV (45.6 mg) and compound V (30.2 mg).
Example 3 structural identification of Compounds
Hydrogen spectra of Compounds I and II 1 H-NMR) and carbon Spectrometry 13 C-NMR) nuclear magnetic data are shown in Table 1:
TABLE 1 Compounds I and II 1 H-NMR 13 Nuclear magnetic data of C-NMR (pyridine-d) 5 )
Hydrogen spectra of Compounds III, IV and V 1 H-NMR) and carbon Spectrometry 13 C-NMR) nuclear magnetic data are shown in Table 2:
TABLE 2 Compounds III, IV and V 1 H-NMR 13 Nuclear magnetic data (CDCl) of C-NMR 3 )
m represents multiple peaks or overlaps with other signals.
The physicochemical properties of the compound shown in the formula I, the compound shown in the formula II, the compound shown in the formula III, the compound shown in the formula IV and the compound shown in the formula V are as follows:
compound I (ileosposide A)
White powder, positive for Liebermann-Burchard reaction and positive for Molish reaction. Molecular formula C 41 H 65 O 16 SNa. ESI-MS gives m/z 869[ M+H ]] + ,m/z 891[M+Na] + ,m/z 845[M-Na] - The method comprises the steps of carrying out a first treatment on the surface of the HR-ESI-MS gives m/z 845.3983[ M-Na ]] - (C 41 H 65 O 16 S, calculated as 845.3993).
Compound II (ileosposide B)
White powder, positive for Liebermann-Burchard reaction and positive for Molish reaction. Molecular formula C 41 H 65 O 16 SNa. ESI-MS gives m/z 869[ M+H ]] + ,m/z 891[M+Na] + ,m/z 845[M-Na] - The method comprises the steps of carrying out a first treatment on the surface of the HR-ESI-MS gives m/z 845.3946[ M-Na ]] - (C 41 H 65 O 16 S, calculated as 845.3993).
Compound III 4-methyl-11 alpha, 12 alpha-epoxy-2-hydroxy-3, 5-dioxarsa-1, 4-dine-28-oic acid gamma-lactone
White powder. ESI-MS (negative) gives m/z 479.3[ M-H ]] - HR-ESI-MS gives M/z481.2594[ M+H ]] + (C 29 H 37 O 6 Calculated as 481.2590) to determine the molecular formula of the compound as C 29 H 36 O 6 。
Compounds IV 4-methyl-11 alpha, 12 alpha-epoxy-2-hydroxy-3, 5-dioxa-1,4,20 (29) -trie-28-oic acid gamma-lactone
White powder. ESI-MS (negative) gives m/z 476.9[ M-H ]] - The molecular weight of this compound was assumed to be 478.
Compound V11 alpha, 12 alpha-epoxy-3 beta-hydroxy-24-norarsa-4 (23) -en-28,13 beta-olide
White powder. ESI-MS (negative) gives m/z 455.0[ M+H ]] + HR-ESI-MS gives m/z 477.2971[ M+Na ]] + (C 29 H 42 NaO 4 Calculated as 477.2981) to determine the molecular formula of the compound as C 29 H 42 O 4 。
Anti-inflammatory Activity of the Compounds of example 4
Test cells: mouse mononuclear macrophage RAW264.7 was purchased from Shanghai cell bank of China academy of sciences and cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37deg.C and 5% CO 2 Culturing in an incubator, and when the cell area in the culture dish reaches 70% -80% of the culture dish, carrying out passage, wherein the specific operation is as follows: first, the cells in the petri dish were all collected into a 50mL sterile centrifuge tube, and then placed in a low temperature centrifuge (4 ℃) and centrifuged at 1000rpm for 3min. Removing the supernatant after centrifugation, adding new RPMI-1640 culture solution to gently blow the bottom cells, forming uniform cell solution, transferring into new culture dishes, and placing into an incubator for continuous culture. The cells in logarithmic growth phase are taken in the experiment.
Sample solution to be tested: the compounds I and II prepared in example 2 were dissolved in cell culture grade DMSO to prepare stock solutions at a concentration of 50mM, and stored in a-25℃refrigerator until use, and the stock solutions were diluted to the concentrations used with cell culture grade DMSO.
Cytotoxicity evaluation: MTT solution (final concentration 200. Mu.g/mL) was added to each well of the above-mentioned culture plate, and after culturing in a 5% carbon dioxide incubator for 4 hours, the supernatant was discarded, the residual liquid was dried by suction, 150. Mu.L of DMSO was added, and after shaking for 10 minutes to sufficiently dissolve the formazan crystals formed, the absorbance was measured at 570nm with 630nm as a reference wavelength.
PGE 2 Determination of the release amount: macrophage cell line from mice in RPMI 1640 mediumRAW264.7 was diluted to 5X 10 5 cells/mL, seeded in 96-well cell culture plates, 200. Mu.L of cell suspension was added per well. CO 2 After 1h incubation in the incubator, LPS (final concentration 1. Mu.g/mL) and test samples of different concentrations were added to each well, and LPS group (no sample to be tested) and blank group (equal volume of DMSO) were set at the same time, 4 parallel wells per sample. At 37 ℃ CO 2 After culturing in a constant temperature incubator for 24 hours, 100. Mu.L of the culture supernatant was aspirated, and the measurement was performed according to the kit instructions. PGE (PGE) 2 The higher the inhibition, the stronger the anti-inflammatory activity of the compound.
Cytotoxicity of the compound:
cell viability of the compounds of table 3 against RAW264.7
Induction of PGE release from RAW264.7 cells by lipopolysaccharide 2 Is effective in inhibiting activity of (a)
TABLE 4 PGE release on RAW264.7 cells 2 Is effective in inhibiting activity of (a)
Investigation of PGE release induced by Compounds I and II on lipopolysaccharide RAW264.7 cells 2 Is selected as a positive drug. The test results show that compounds I and II show significantly greater activity than the positive drug, hydrocortisone, and are not toxic.
Example 5 anti-inflammatory mechanism of Compounds
Protein extraction was used to determine iNOS and COX-2: taking mouse macrophage RAW264.7 in logarithmic phase, diluting to 1×10 6 The single cell suspension was inoculated into a 60mm×15mm dish and incubated at 37℃for 1h. After the cells had adhered to the wall, the cells were divided into three groups, namely, a blank group, an LPS group, and an administration group. LPS group was added with 1g/mL LPS, and the drug administration groups were added with different concentrations of IA-C9E6-4 and IA-C9D4a (final)Concentrations were 12.5, 25, 50, 100. Mu.M) and 1g/mL LPS, respectively. After incubation for 24h, the supernatants of each group of cells were discarded, the reaction was stopped with PBS and the cells were washed, 1mL of PBS was added, and the cells were collected with a cell scraper in a 1.5mL centrifuge tube. After centrifugation at 13000r/min for 6min, the supernatant was removed as much as possible, 60. Mu.L of PBS containing 1% PMSF was added, and the tube was placed in an ice-water bath with an ultrasonic breaker to carry out cell disruption. After centrifugation at 13000r/min for 6min, the supernatant was aspirated into a 0.5mL centrifuge tube and the protein concentration was determined by the Bradford method using the kit. Adding 5 Xprotein loading buffer solution into a 0.5mL centrifuge tube according to the ratio of 4:1, placing in a boiling water bath for 5min to denature protein, and storing in a refrigerator at-20deg.C for use.
Protein extraction was used to determine IκB- α: taking mouse macrophage RAW264.7 in logarithmic phase, diluting to 1×10 6 The single cell suspension was inoculated into a 60mm×15mm dish and incubated at 37℃for 24 hours. After 24h, the cells were divided into three groups, namely, blank group, LPS group, and dosing group. The IA-C9E6-4 and IA-C9D4a (final concentration is 12.5, 25, 50 and 100. Mu.M) with different concentrations are respectively added into the drug administration group, and after the culture is continued for 1h, 1g/mL LPS is added into the drug administration group and LPS group for 10min. The supernatant of each group of cells is respectively discarded, 1mL of PBS is added to stop the reaction and wash the cells, 1mL of PBS is added, and the cells are collected in a 1.5mL centrifuge tube by a cell scraper; after centrifugation at 13000r/min for 6min, the supernatant was removed as much as possible, 60. Mu.L of PBS containing 1% PMSF was added, and the tube was placed in an ice-water bath with an ultrasonic breaker to carry out cell disruption. After centrifugation at 13000r/min for 6min, the supernatant was aspirated into a 0.5mL centrifuge tube and the protein concentration was determined by the Bradford method using the kit. Adding 5 Xprotein loading buffer solution into a 0.5mL centrifuge tube according to the ratio of 4:1, placing in a boiling water bath for 5min to denature protein, and storing in a refrigerator at-20deg.C for use.
Protein extraction was used to determine ERK and p-ERK: taking mouse macrophage RAW264.7 in logarithmic phase, diluting to 1×10 6 The single cell suspension was inoculated into a 60mm×15mm dish and incubated at 37℃for 24 hours. After 24h, the cells were divided into three groups, namely, blank group, LPS group, and dosing group. The medicine is added with different concentrations respectivelyIA-C9E6-4 and IA-C9D4a (final concentrations of 12.5, 25, 50, 100. Mu.M, respectively) were cultured for 1 hour, and after further culturing, 1g/mL LPS was added to the administration group and LPS group for 1 hour. The supernatant of each group of cells is respectively discarded, 1mL of PBS is added to stop the reaction and wash the cells, 1mL of PBS is added, and the cells are collected in a 1.5mL centrifuge tube by a cell scraper; after centrifugation at 13000r/min for 6min, the supernatant was removed as much as possible, 60. Mu.L of PBS containing 1% PMSF was added, and the tube was placed in an ice-water bath with an ultrasonic breaker to carry out cell disruption. After centrifugation at 13000r/min for 6min, the supernatant was aspirated into a 0.5mL centrifuge tube and the protein concentration was determined by the Bradford method using the kit. Adding 5 Xprotein loading buffer solution into a 0.5mL centrifuge tube according to the ratio of 4:1, placing in a boiling water bath for 5min to denature protein, and storing in a refrigerator at-20deg.C for use.
Western Blot: after gel electrophoresis of 30g of protein, transfer to nitrocellulose membrane, seal the membrane in 5% nonfat milk powder diluted in PBS buffer for 4 hours at room temperature. After washing the membranes three times with TBST (20 mM Tris-HCl,150mM NaCl,0.05% Tween 20), the membranes were incubated overnight at 4℃with the corresponding primary antibody solutions (anti-iNOS, anti-COX-2, anti-IκB- α, anti-phosphorylated ERK, anti-phosphorylated JNK, anti-phosphorylated p38, anti-non-phosphorylated ERK, anti-non-phosphorylated JNK, anti-non-phosphorylated p 38). After washing the membrane three times with TBST, the secondary antibody labeled with horseradish peroxidase was incubated at room temperature for 1 hour. Membranes were washed three times with TBST, western blotted with chemiluminescent kit (ECL) and exposed to film. Images were collected and the proteins were quantified by densitometric analysis of the iNOS, COX-2, ikb- α, ERK, JNK, p38, phospho-ERK, phospho-JNK, phospho-p38 bands corresponding to the proteins using band 4.0 software. The data obtained after band analysis of iNOS, COX-2, ikb- α protein was treated with β actin as an internal control, and the data obtained after band analysis of phospho-ERK, phospho-JNK, phospho-P38 protein was treated with ERK, JNK, P as a control, giving a relative protein content, and the final values were expressed as mean ± s.d. with T-test, P <0.05 being considered significant differences in group comparisons.
The test results are shown in FIGS. 1-3.
The results show that:
the high concentration of the compound II has an inhibiting effect on the high expression of the iNOS and COX-2 proteins of the RAW264.7 cells induced by LPS, while the compound I has no obvious inhibiting effect on the high expression of the iNOS and COX-2 proteins of the RAW264.7 cells induced by LPS.
The high concentration of the compound I and the low, medium and high concentrations of the compound II have an inhibiting effect on LPS-induced degradation of RAW264.7 cell IκB-alpha protein.
Compounds I and II are able to block the phosphorylation of MAPK/ERK proteins and MAPK/p38 proteins, but have less effect on the phosphorylation of MAPK/JNK proteins. The MAPK signal path has regulation and control effects on the expression of iNOS and COX-2 proteins, and the compounds further down regulate the expression of the iNOS and COX-2 proteins by blocking the MAPK/ERK/p38 signal path, thereby playing an anti-inflammatory role.
Claims (8)
1. A compound of formula i or II or a pharmaceutically acceptable salt thereof;
。
2. a process for the preparation of a compound of formula I or II according to claim 1, characterized in that,
(1) Extracting dry root of roughhaired holly with ethanol, concentrating to obtain total extract;
(2) Suspending the total extract with water, loading onto macroporous adsorbent resin column, and gradient eluting with 0%,30%, 50%, 70%, 95% ethanol to obtain five fractions of IA-A, IA-B, IA-C, IA-D, IA-E;
(3) Separating IA-C by silica gel column chromatography, and performing gradient elution by using a dichloromethane/chloroform-methanol system of 100:0, 99:1, 98:2, 97:3, 95:5, 93:7,9:1,8:2,7:3,6:4,1:1,0:100 to obtain a fraction IA-C1-IA-C12 respectively;
(4) Separating IA-C9 by ODS column chromatography, and gradient eluting with 10%,20%,30%,40%,50%,60%,70%,80%, and 100% methanol-water system to obtain IA-C9-A-IA-C9-I;
(5) Separating IA-C9-D by silica gel column chromatography, and performing gradient elution by using a methylene dichloride-methanol-water system 9:1:0.1, a methylene dichloride-methanol-water system 8:2:0.2, a methylene dichloride-methanol-water system 7:3:0.5 and a methylene dichloride-methanol-water system 6:4:0.8 to obtain IA-C9-D1-IA-C9-D4 respectively;
IA-C9-E was separated by silica gel column chromatography using methylene chloride-methanol-water system: sequentially performing gradient elution on dichloromethane-methanol-water according to the volume ratio of 9:1:0.1,8:2:0.2,7:3:0.5 and 6:4:0.8 to obtain IA-C9-E1-IA-C9-E4 respectively;
(6) Crystallizing IA-C9-D4 in methanol to obtain a compound II; the IA-C9-E3 is further separated to prepare a high performance liquid phase to obtain the compound I, wherein the mobile phase is an acetonitrile-water solution with the concentration of 10-20%.
3. The process according to claim 2, wherein in step (1), the ethanol is used in a volume concentration of 60 to 90% and the extraction is performed by heating and refluxing for 1 to 3 times, each for 1 to 3 hours.
4. A pharmaceutical composition comprising one or more of the compounds of formula i or II as defined in claim 1 or a pharmaceutically acceptable salt thereof.
5. A pharmaceutical composition comprising one or more of the compounds of formula i or II as defined in claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.
6. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition is in the form of an injection, a tablet, a powder, a granule, a pill, a capsule, an oral liquid, a paste, a cream or a spray.
7. Use of a compound of formula i or II as defined in claim 1 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition as defined in claim 4 or 5 for the manufacture of a medicament for the treatment and/or prophylaxis of inflammation.
8. The use of claim 7, wherein the inflammation is induced by lipopolysaccharide in RAW264.7 cellsPGE release 2 Inflammation caused by the above-mentioned drugs.
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| Anti-inflammatory triterpene glycosides from the roots of Ilex dunniana Levl;Yu-Sheng Shi 等;Molecules;第22卷(第7期);摘要，图1，表2-4 * |
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