CN115011640A - Application and method for preparing compound by using monascus durissus A2 - Google Patents
Application and method for preparing compound by using monascus durissus A2 Download PDFInfo
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- CN115011640A CN115011640A CN202210694577.1A CN202210694577A CN115011640A CN 115011640 A CN115011640 A CN 115011640A CN 202210694577 A CN202210694577 A CN 202210694577A CN 115011640 A CN115011640 A CN 115011640A
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- silica gel
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 123
- 238000000034 method Methods 0.000 title claims abstract description 35
- 241000228347 Monascus <ascomycete fungus> Species 0.000 title abstract description 11
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000003814 drug Substances 0.000 claims abstract description 10
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 5
- 210000002540 macrophage Anatomy 0.000 claims abstract description 4
- 238000010828 elution Methods 0.000 claims description 105
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 96
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 84
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 63
- 238000000855 fermentation Methods 0.000 claims description 59
- 230000004151 fermentation Effects 0.000 claims description 58
- 239000003480 eluent Substances 0.000 claims description 53
- 238000010829 isocratic elution Methods 0.000 claims description 51
- 238000010898 silica gel chromatography Methods 0.000 claims description 51
- 239000000287 crude extract Substances 0.000 claims description 31
- 238000004809 thin layer chromatography Methods 0.000 claims description 29
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 26
- 239000011259 mixed solution Substances 0.000 claims description 26
- 239000000203 mixture Substances 0.000 claims description 26
- 239000003208 petroleum Substances 0.000 claims description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 24
- 239000001963 growth medium Substances 0.000 claims description 23
- 244000113306 Monascus purpureus Species 0.000 claims description 20
- 235000002322 Monascus purpureus Nutrition 0.000 claims description 20
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- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 19
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- 150000003839 salts Chemical class 0.000 claims description 9
- QFLWZFQWSBQYPS-AWRAUJHKSA-N (3S)-3-[[(2S)-2-[[(2S)-2-[5-[(3aS,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]-3-methylbutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-4-[1-bis(4-chlorophenoxy)phosphorylbutylamino]-4-oxobutanoic acid Chemical compound CCCC(NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@@H](NC(=O)CCCCC1SC[C@@H]2NC(=O)N[C@H]12)C(C)C)P(=O)(Oc1ccc(Cl)cc1)Oc1ccc(Cl)cc1 QFLWZFQWSBQYPS-AWRAUJHKSA-N 0.000 claims description 8
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- AICOOMRHRUFYCM-ZRRPKQBOSA-N oxazine, 1 Chemical compound C([C@@H]1[C@H](C(C[C@]2(C)[C@@H]([C@H](C)N(C)C)[C@H](O)C[C@]21C)=O)CC1=CC2)C[C@H]1[C@@]1(C)[C@H]2N=C(C(C)C)OC1 AICOOMRHRUFYCM-ZRRPKQBOSA-N 0.000 claims description 4
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- HVBSAKJJOYLTQU-UHFFFAOYSA-N 4-aminobenzenesulfonic acid Chemical compound NC1=CC=C(S(O)(=O)=O)C=C1 HVBSAKJJOYLTQU-UHFFFAOYSA-N 0.000 description 2
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- MCSXGCZMEPXKIW-UHFFFAOYSA-N 3-hydroxy-4-[(4-methyl-2-nitrophenyl)diazenyl]-N-(3-nitrophenyl)naphthalene-2-carboxamide Chemical compound Cc1ccc(N=Nc2c(O)c(cc3ccccc23)C(=O)Nc2cccc(c2)[N+]([O-])=O)c(c1)[N+]([O-])=O MCSXGCZMEPXKIW-UHFFFAOYSA-N 0.000 description 1
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- VILAVOFMIJHSJA-UHFFFAOYSA-N dicarbon monoxide Chemical group [C]=C=O VILAVOFMIJHSJA-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
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- A61K31/19—Carboxylic acids, e.g. valproic acid
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- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
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- C07C39/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C41/00—Preparation of ethers; Preparation of compounds having groups, groups or groups
- C07C41/01—Preparation of ethers
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- C07C43/00—Ethers; Compounds having groups, groups or groups
- C07C43/02—Ethers
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
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- C07C49/255—Unsaturated compounds containing keto groups bound to acyclic carbon atoms containing ether groups, groups, groups, or groups
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- C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
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- C07C49/713—Unsaturated compounds containing a keto groups being part of a ring containing hydroxy groups a keto group being part of a six-membered ring
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- C—CHEMISTRY; METALLURGY
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
- C07C51/47—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C65/00—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
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Abstract
The invention provides a method and application of monascus granatum A2(Aspergillus puniceus A2) in preparation of various compounds and application of the compounds in preparation of a medicine for inhibiting macrophages from releasing nitric oxide, and belongs to the field of marine microbial medicines. The method can prepare various compounds with pharmacological activity, and the preparation method has the advantages of simple substrate, simple and convenient operation method and abundant products. The obtained compound has good activity of inhibiting macrophage from releasing nitric oxide, and has good anti-inflammatory activity.
Description
Technical Field
The invention relates to the field of marine microbial medicines, and relates to application and a method for preparing a compound by using monascus granatum A2.
Background
By adapting to extreme environmental conditions, deep-sea microorganisms have unique physiological metabolic processes, and further show diversity, novelty and remarkable biological activity of the chemical structures of metabolites of the deep-sea microorganisms. The marine microorganisms are one of the most abundant sources of pharmacological active natural products at present, and the bioactive compounds not only can help deep-sea fungi to defend natural enemies in a natural ecosystem, but also have the potential of becoming human diseases for treatment and new biological targets for search, and provide important support for marine medicine research and development.
In the prior art, the number of marine microorganisms is estimated to be 0.1 to 2 hundred million due to the wide variety of marine microorganisms. To date, about 150 or more thousands of microorganisms have been found in humans, and the rest exist in the ocean except for 7.2 thousands. Although the metabolites of some marine microorganisms have pharmacological active substances, not all marine microorganisms can be fermented and cultured to obtain active substances, and the marine microorganisms are used for fermentation, fermentation and culture, and separation and purification, so that the active substances with high yield and high purity can be obtained.
Therefore, a preparation method which is simple to operate, rich in products and capable of preparing various compounds with pharmacological activity is still needed.
Disclosure of Invention
In order to solve the above problems, the present invention provides a method for preparing a compound. The invention utilizes a solid fermentation method to carry out large-scale fermentation culture on a strain of monascus durianus A2(Aspergillus puniceus A2), and adopts a plurality of chromatographic separation and purification methods to separate and obtain a compound from a fermentation product. And then, the anti-inflammatory activity of the monomeric compound is tested through indexes such as cell activity, inflammatory mediators and the like, and reference is provided for exploring the application of the active compound in the pharmaceutical industry.
The invention elaborates and adopts rice solid culture medium to ferment and culture a monascus granatum A2(Aspergillus puniceus A2), comprehensively uses a plurality of chromatographic separation technologies such as silica gel column chromatography, sephadex column chromatography, high performance liquid chromatography and the like to separate and purify the fermentation product, and then uses modern spectrum technologies such as NMR, mass spectrum and the like to identify 11 monomeric compounds including 2 new compounds by combining with literature data reports; the 11 compounds were tested for anti-inflammatory activity by anti-inflammatory activity assay. The research result is expected to provide important reference for the application of novel active natural products in the fields of medicine and food.
In a first aspect, the present invention provides the use of Aspergillus pungens a2(Aspergillus puniceus a2) for the preparation of a compound.
In some embodiments, use of Aspergillus niger A2(Aspergillus puniceus A2) for the preparation of at least one compound,
said monascus granatum a2(Aspergillus puniceus) is any monascus granatum a2 disclosed. In some embodiments, the monascus purpureus a2(Aspergillus puniceus a2) may be monascus purpureus a2(Aspergillus puniceus a2) isolated from sediments deep in the pacific (156 ° 31'20"E,12 ° 27'7" N)4841 m. The ITS gene sequence analysis of said Aspergillus niger A2(Aspergillus puniceus A2) isolated from sediments deep in the Pacific (156 ℃ 31'20"E,12 ℃ 27'7" N)4841m showed a degree of similarity of 99.83% to Aspergillus puniceus strain SRRC 2155, which was identified as Aspergillus puniceus A2; the sequencing results of the Monascus purpureus solani A2(Aspergillus puniceus A2) isolated from sediments deep in the Pacific (156 ℃ 31'20"E,12 ℃ 27'7" N)4841m were submitted to GenBank under accession number OM 063154. The monascus purpureus a2(Aspergillus puniceus a2) isolated from sediments in the depth of 4841m in the pacific (156 ° 31'20"E,12 ° 27'7" N) was deposited at the center of innovation of engineering and technology for the development and utilization of marine biological resources of the third marine institute of natural resources.
In a second aspect, the present invention provides a method of preparing a compound.
In some embodiments, a method of making a compound, comprises:
step S1: carrying out fermentation culture on monascus purpureus A2(Aspergillus puniceus A2) in a culture medium to obtain a fermentation product; then, the user can use the device to perform the operation,
step S2: and (4) extracting the fermentation product obtained in the step S1 to obtain a crude extract, and separating and purifying the crude extract to obtain a compound. The monascus granatum A2(Aspergillus puniceus A2) provided by the invention is adopted for biosynthesis, which is beneficial to preparing various compounds with anti-inflammatory effects, and the method is simple to operate and the obtained compounds are high in yield.
In some embodiments, the compound comprises at least one of the following compounds:
in some embodiments, the compound comprises compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8, compound 9, compound 10, and compound 11.
In some embodiments, the crude extract may be concentrated prior to isolation and purification.
In some embodiments, the concentration may be rotary evaporation concentration or concentration under reduced pressure.
In some embodiments, the culture medium may include rice, sodium chloride, and water.
In some embodiments, the sodium chloride is sea salt.
In some embodiments, the rice may be included in an amount of 40.0 wt% to 45.0 wt%, the sodium chloride may be included in an amount of 1.5 wt% to 2.0 wt%, and the balance may be water, based on the total mass of the medium. In some embodiments, the rice is present in an amount of 42.5 wt%, the sodium chloride is present in an amount of 1.8 wt%, and the balance is water, based on the total mass of the medium.
In some embodiments, the temperature of the fermentation culture is between 25 ℃ and 28 ℃. In some embodiments, the fermentation culture is maintained at a temperature of 25 deg.C
In some embodiments, the fermentation culture is between 42 days and 55 days. In some embodiments, the fermentation culture is for a culture time of 50 days.
In some embodiments, the extraction may comprise extraction with ethyl acetate.
In some embodiments, the step S2 of separating and purifying comprises: performing gradient elution a by normal phase silica gel column chromatography, wherein the gradient elution a is performed by using a volume ratio of 8-10 times of column volume of 1: 0-3: 1 petroleum ether: ethyl acetate mixture was subjected to gradient elution and detected using Thin Layer Chromatography (TLC); combining similar components to obtain 2 components, and respectively naming the components as a component Fr.1 and a component Fr.2 according to the order of peak appearance of eluent.
In some embodiments, the component fr.1 may be subjected to a gradient elution b over a reverse phase silica gel column, which may be a volume ratio of 9: 11-1: 0 methanol: gradient elution is carried out on the water mixed solution to obtain 2 components which are respectively named as a component Fr.1.1 and a component Fr.1.2 according to the order of peak appearance of the eluent.
In some embodiments, the component fr.1.1 may be isocratic eluted through normal phase silica gel column chromatography, which may be 3: 1 petroleum ether: eluting with ethyl acetate mixture to obtain 3 components, and respectively naming the components as Fr.1.1.1, Fr.1.1.2 and Fr.1.1.3 according to the order of peak appearance of the eluent.
In some embodiments, the component fr.1.1.1 may be isocratic eluted through normal phase silica gel column chromatography, which isocratic eluted d may be, using a volume ratio of 10-15 column volumes of 50: 1 dichloromethane: eluting with acetone mixed solution to obtain 4 components, and respectively naming the components as Fr.1.1.1.1.1, Fr.1.1.2, Fr.1.1.1.3 and Fr.1.1.1.4 according to the order of peak appearance of the eluent.
In some embodiments, the component fr.1.1.1.3 may be purified to provide compound 9.
In some embodiments, the component fr.1.1.1.4 may be purified to provide compound 8.
In some embodiments, the component fr.1.1.2 may be isocratic eluted e by normal phase silica gel column chromatography, which may be eluted using 10-15 column volumes of dichloromethane to give 1 component, designated as component fr.1.1.2.1; the fraction fr.1.1.2.1 is purified to give compounds 5 and 6.
In some embodiments, the component fr.1.1.3 may be subjected to isocratic elution f by normal phase silica gel column chromatography, and the isocratic elution f may be performed by using dichloromethane with 10 to 15 times column volume to obtain 2 components which are respectively named as component fr.1.1.3.1 and component fr.1.1.3.2 according to the order of peak appearance of eluent; the fraction fr.1.1.3.1 is purified to give compound 3 and compound 4.
In some embodiments, the component fr.1.2 may elute g via a normal phase silica gel column chromatography gradient, which may be a volume ratio of 400: 1-1: 1 dichloromethane: gradient elution is carried out on the methanol mixed solution to obtain 2 components which are respectively named as a component Fr.1.2.1 and a component Fr.1.2.2 according to the order of peak appearance of the eluent; the fraction fr.1.2.2 can be purified to give compound 7.
In some embodiments, the component fr.2 may be eluted through a reverse phase silica gel column gradient, h, which may be a volume ratio using 5 to 8 column volumes of 3: 7-1: 0 methanol: gradient elution is carried out on the water mixed solution to obtain 3 components which are respectively named as a component Fr.2.1, a component Fr.2.2 and a component Fr.2.3 according to the order of peak appearance of the eluent.
In some embodiments, the fraction fr.2.1 may be eluted isocratically through a sephadex column, which may be eluted using 6-9 column volumes of methanol to give 1 fraction, designated fraction fr.2.1.1; the fraction Fr.2.1.1 is purified to give compounds 10 and 11.
In some embodiments, the component fr.2.2 may be purified to provide compound 2.
In some embodiments, the fraction fr.2.3 may be isocratically eluted through a sephadex column, which isocratic elution j may be an elution with 6-9 column volumes of methanol to give fraction fr.2.3.1, which fraction fr.2.3.1 is purified to give compound 1.
In some embodiments, the "purified form" prior to obtaining each compound may include purification by preparative high performance liquid chromatography or semi-preparative high performance liquid chromatography.
In some embodiments, a method of making compound 1, comprises: step S1: carrying out fermentation culture on monascus purpureus A2(Aspergillus puniceus A2) in a culture medium to obtain a fermentation product; then, the user can use the device to perform the operation,
step S2: extracting the fermentation product obtained in the step S1 to obtain a crude extract, and separating and purifying the crude extract to obtain a compound;
the separation and purification in step S2 may include: and (3) performing gradient elution a by using normal-phase silica gel column chromatography, wherein the gradient elution a is a gradient elution method which uses a volume ratio of 8-10 times of column volume to 1: 0-3: 1 petroleum ether: ethyl acetate mixture was subjected to gradient elution and detected using Thin Layer Chromatography (TLC); combining similar components to obtain 2 components, and respectively naming the components as a component Fr.1 and a component Fr.2 according to the order of peak appearance of eluent, wherein the component Fr.2 can be subjected to gradient elution h through a reverse phase silica gel column, and the gradient elution h can be performed by using a volume ratio of 5-8 times of column volume of 3: 7-1: 0 methanol: gradient elution is carried out on the water mixed solution to obtain 3 components, the components are respectively named as a component Fr.2.1, a component Fr.2.2 and a component Fr.2.3 according to the peak appearance sequence of the eluent, the component Fr.2.3 can be subjected to isocratic elution j through a sephadex column, the isocratic elution j can be eluted by using methanol with 6-9 times of column volume to obtain 1 component, the component Fr.2.3.1 is named as a component Fr.2.3.1, and the component Fr.2.3.1 is purified to obtain a compound 1.
In some embodiments, a method of making compound 2, comprising: step S1: carrying out fermentation culture on monascus purpureus A2(Aspergillus puniceus A2) in a culture medium to obtain a fermentation product; then, the user can use the device to perform the operation,
step S2: extracting the fermentation product obtained in the step S1 to obtain a crude extract, and separating and purifying the crude extract to obtain a compound;
the separation and purification in step S2 may include: performing gradient elution a by normal phase silica gel column chromatography, wherein the gradient elution a is performed by using a volume ratio of 8-10 times of column volume of 1: 0-3: 1 petroleum ether: ethyl acetate mixture was subjected to gradient elution and detected using Thin Layer Chromatography (TLC); combining similar components to obtain 2 components, and respectively naming the components as a component Fr.1 and a component Fr.2 according to the order of peak appearance of eluent, wherein the component Fr.2 can be subjected to gradient elution h through a reverse phase silica gel column, and the gradient elution h can be performed by using a volume ratio of 5-8 times of column volume of 3: 7-1: 0 methanol: gradient elution is carried out on the water mixed solution to obtain 3 components, the components are respectively named as a component Fr.2.1, a component Fr.2.2 and a component Fr.2.3 according to the order of peak appearance of the eluent, and the component Fr.2.2 can be purified to obtain a compound 2.
In some embodiments, a method of making compound 3, comprising: step S1: carrying out fermentation culture on monascus purpureus A2(Aspergillus puniceus A2) in a culture medium to obtain a fermentation product; then, the user can use the device to perform the operation,
step S2: extracting the fermentation product obtained in the step S1 to obtain a crude extract, and separating and purifying the crude extract to obtain a compound;
the separation and purification in step S2 may include: performing gradient elution a by normal phase silica gel column chromatography, wherein the gradient elution a is performed by using a volume ratio of 8-10 times of column volume of 1: 0-3: 1 petroleum ether: ethyl acetate mixture was subjected to gradient elution and detected using Thin Layer Chromatography (TLC); combining similar components to obtain 2 components, and respectively naming the components as a component Fr.1 and a component Fr.2 according to the order of peak appearance of eluent, wherein the component Fr.1 can be subjected to gradient elution b by a reverse phase silica gel column, and the gradient elution b can be performed by using a volume ratio of 5-8 times of column volume of 9: 11-1: 0 methanol: gradient elution is carried out on the water mixed solution to obtain 2 components which are respectively named as a component Fr.1.1 and a component Fr.1.2 according to the order of peak appearance of the eluent; the fraction fr.1.1 may be isocratic eluted through normal phase silica gel column chromatography, the isocratic elution c may be a volumetric ratio of 3: 1 petroleum ether: eluting with ethyl acetate mixture to obtain 3 components, and respectively naming the components as Fr.1.1.1, Fr.1.1.2 and Fr.1.1.3 according to the order of peak appearance of the eluate; the component Fr.1.1.3 can be subjected to isocratic elution f by normal-phase silica gel column chromatography, the isocratic elution f can be eluted by using dichloromethane with 10-15 times column volume to obtain 2 components, and the components are respectively named as a component Fr.1.1.3.1 and a component Fr.1.1.3.2 according to the order of peak appearance of eluent; the fraction fr.1.1.3.1 is purified to give compound 3.
In some embodiments, a method of making compound 4, comprising: step S1: carrying out fermentation culture on monascus purpureus A2(Aspergillus puniceus A2) in a culture medium to obtain a fermentation product; then, the user can use the device to perform the operation,
step S2: extracting the fermentation product obtained in the step S1 to obtain a crude extract, and separating and purifying the crude extract to obtain a compound;
the separation and purification in step S2 may include: performing gradient elution a by normal phase silica gel column chromatography, wherein the gradient elution a is performed by using a volume ratio of 8-10 times of column volume of 1: 0-3: 1 petroleum ether: ethyl acetate mixture was subjected to gradient elution and detected using Thin Layer Chromatography (TLC); combining similar components to obtain 2 components, and respectively naming the components as a component Fr.1 and a component Fr.2 according to the order of peak appearance of eluent, wherein the component Fr.1 can be subjected to gradient elution b by a reverse phase silica gel column, and the gradient elution b can be performed by using a volume ratio of 5-8 times of column volume of 9: 11-1: 0 methanol: gradient elution is carried out on the water mixed solution to obtain 2 components which are respectively named as a component Fr.1.1 and a component Fr.1.2 according to the order of peak appearance of the eluent; the fraction fr.1.1 may be isocratic eluted through normal phase silica gel column chromatography, the isocratic elution c may be a volumetric ratio of 3: 1 petroleum ether: eluting with ethyl acetate mixture to obtain 3 components, and respectively naming the components as Fr.1.1.1, Fr.1.1.2 and Fr.1.1.3 according to the order of peak appearance of the eluate; the component Fr.1.1.3 can be subjected to isocratic elution f by normal-phase silica gel column chromatography, the isocratic elution f can be eluted by using dichloromethane with 10-15 times column volume to obtain 2 components, and the components are respectively named as a component Fr.1.1.3.1 and a component Fr.1.1.3.2 according to the order of peak appearance of eluent; the fraction Fr.1.1.3.1 is purified to give compound 4.
In some embodiments, a method of making compound 5, comprising: step S1: carrying out fermentation culture on monascus purpureus A2(Aspergillus puniceus A2) in a culture medium to obtain a fermentation product; then, the user can use the device to perform the operation,
step S2: extracting the fermentation product obtained in the step S1 to obtain a crude extract, and separating and purifying the crude extract to obtain a compound;
the separation and purification in step S2 may include: performing gradient elution a by normal phase silica gel column chromatography, wherein the gradient elution a is performed by using a volume ratio of 8-10 times of column volume of 1: 0-3: 1 petroleum ether: ethyl acetate mixture was subjected to gradient elution and detected using Thin Layer Chromatography (TLC); and (2) combining similar components to obtain 2 components, and respectively naming the components as a component Fr.1 and a component Fr.2 according to the order of peak appearance of eluent, wherein the component Fr.1 can be subjected to gradient elution b through a reverse phase silica gel column, and the gradient elution b can be performed by using a volume ratio of 5-8 times of column volume of 9: 11-1: 0 methanol: gradient elution is carried out on the water mixed solution to obtain 2 components which are respectively named as a component Fr.1.1 and a component Fr.1.2 according to the order of peak appearance of the eluent; the fraction fr.1.1 may be isocratic eluted through normal phase silica gel column chromatography, the isocratic elution c may be a volumetric ratio of 3: 1 petroleum ether: eluting with ethyl acetate mixture to obtain 3 components, and respectively naming the components as Fr.1.1.1, Fr.1.1.2 and Fr.1.1.3 according to the order of peak appearance of the eluate; the component Fr.1.1.2 can be subjected to isocratic elution e through normal-phase silica gel column chromatography, and the isocratic elution e can be eluted by using dichloromethane with 10-15 times column volume to obtain 1 component which is named as component Fr.1.1.2.1; the fraction Fr.1.1.2.1 was purified to give compound 5.
In some embodiments, a method of making compound 6, comprising: step S1: carrying out fermentation culture on monascus purpureus A2(Aspergillus puniceus A2) in a culture medium to obtain a fermentation product; then, the user can use the device to perform the operation,
step S2: extracting the fermentation product obtained in the step S1 to obtain a crude extract, and separating and purifying the crude extract to obtain a compound;
the separation and purification in step S2 may include: performing gradient elution a by normal phase silica gel column chromatography, wherein the gradient elution a is performed by using a volume ratio of 8-10 times of column volume of 1: 0-3: 1 petroleum ether: ethyl acetate mixture was subjected to gradient elution and detected using Thin Layer Chromatography (TLC); and (2) combining similar components to obtain 2 components, and respectively naming the components as a component Fr.1 and a component Fr.2 according to the order of peak appearance of eluent, wherein the component Fr.1 can be subjected to gradient elution b through a reverse phase silica gel column, and the gradient elution b can be performed by using a volume ratio of 5-8 times of column volume of 9: 11-1: 0 methanol: gradient elution is carried out on the water mixed solution to obtain 2 components which are respectively named as a component Fr.1.1 and a component Fr.1.2 according to the order of peak appearance of the eluent; the fraction fr.1.1 may be isocratic eluted through normal phase silica gel column chromatography, the isocratic elution c may be a volumetric ratio of 3: 1 petroleum ether: eluting with ethyl acetate mixture to obtain 3 components, and respectively naming the components as Fr.1.1.1, Fr.1.1.2 and Fr.1.1.3 according to the order of peak appearance of the eluate; the component Fr.1.1.2 can be subjected to isocratic elution e through normal-phase silica gel column chromatography, and the isocratic elution e can be eluted by using dichloromethane with 10-15 times column volume to obtain 1 component which is named as component Fr.1.1.2.1; the fraction fr.1.1.2.1 is purified to give compound 6.
In some embodiments, a method of making compound 7, comprising: step S1: carrying out fermentation culture on monascus purpureus A2(Aspergillus puniceus A2) in a culture medium to obtain a fermentation product; then, the user can use the device to perform the operation,
step S2: extracting the fermentation product obtained in the step S1 to obtain a crude extract, and separating and purifying the crude extract to obtain a compound;
the separation and purification in step S2 may include: performing gradient elution a by normal phase silica gel column chromatography, wherein the gradient elution a is performed by using a volume ratio of 8-10 times of column volume of 1: 0-3: 1 petroleum ether: ethyl acetate mixture was subjected to gradient elution and detected using Thin Layer Chromatography (TLC); combining similar components to obtain 2 components, and respectively naming the components as a component Fr.1 and a component Fr.2 according to the order of peak appearance of eluent, wherein the component Fr.1 can be subjected to gradient elution b by a reverse phase silica gel column, and the gradient elution b can be performed by using a volume ratio of 5-8 times of column volume of 9: 11-1: 0 methanol: gradient elution is carried out on the water mixed solution to obtain 2 components which are respectively named as a component Fr.1.1 and a component Fr.1.2 according to the order of peak appearance of the eluent; the component fr.1.2 may be subjected to a normal phase silica gel column chromatography gradient elution g, which may be a gradient elution using a volume ratio of 8-10 column volumes of 400: 1-1: 1 dichloromethane: gradient elution is carried out on the methanol mixed solution to obtain 2 components which are respectively named as a component Fr.1.2.1 and a component Fr.1.2.2 according to the order of peak appearance of the eluent; the fraction fr.1.2.2 can be purified to give compound 7.
In some embodiments, a method of making compound 8, comprising: step S1: carrying out fermentation culture on monascus purpureus A2(Aspergillus puniceus A2) in a culture medium to obtain a fermentation product; then, the user can use the device to perform the operation,
step S2: extracting the fermentation product obtained in the step S1 to obtain a crude extract, and separating and purifying the crude extract to obtain a compound;
the separation and purification in step S2 may include: performing gradient elution a by normal phase silica gel column chromatography, wherein the gradient elution a is performed by using a volume ratio of 8-10 times of column volume of 1: 0-3: 1 petroleum ether: ethyl acetate mixture was subjected to gradient elution and detected using Thin Layer Chromatography (TLC); combining similar components to obtain 2 components, and respectively naming the components as a component Fr.1 and a component Fr.2 according to the order of peak appearance of eluent, wherein the component Fr.1 can be subjected to gradient elution b by a reverse phase silica gel column, and the gradient elution b can be performed by using a volume ratio of 5-8 times of column volume of 9: 11-1: 0 methanol: gradient elution is carried out on the water mixed solution to obtain 2 components which are respectively named as a component Fr.1.1 and a component Fr.1.2 according to the order of peak appearance of the eluent; the fraction fr.1.1 may be isocratic eluted through normal phase silica gel column chromatography, the isocratic elution c may be a volumetric ratio of 3: 1 petroleum ether: eluting with ethyl acetate mixture to obtain 3 components, and respectively naming the components as Fr.1.1.1, Fr.1.1.2 and Fr.1.1.3 according to the order of peak appearance of the eluate; the fraction fr.1.1.1 may be isocratic eluted through normal phase silica gel column chromatography, the isocratic elution d may be a volume ratio of 50: 1 dichloromethane: eluting with acetone mixed solution to obtain 4 components, and respectively naming the components as Fr.1.1.1.1, Fr.1.1.1.2, Fr.1.1.1.3 and Fr.1.1.1.4 according to the order of peak appearance of the eluent; the component Fr.1.1.1.4 can be purified to give compound 8.
In some embodiments, a method of making compound 9, comprising: step S1: carrying out fermentation culture on monascus purpureus A2(Aspergillus puniceus A2) in a culture medium to obtain a fermentation product; then, the user can use the device to perform the operation,
step S2: extracting the fermentation product obtained in the step S1 to obtain a crude extract, and separating and purifying the crude extract to obtain a compound;
the separation and purification in step S2 may include: the separation and purification in step S2 may include: performing gradient elution a by normal phase silica gel column chromatography, wherein the gradient elution a is performed by using a volume ratio of 8-10 times of column volume of 1: 0-3: 1 petroleum ether: ethyl acetate mixture was subjected to gradient elution and detected using Thin Layer Chromatography (TLC); combining similar components to obtain 2 components, and respectively naming the components as a component Fr.1 and a component Fr.2 according to the order of peak appearance of eluent, wherein the component Fr.1 can be subjected to gradient elution b by a reverse phase silica gel column, and the gradient elution b can be performed by using a volume ratio of 5-8 times of column volume of 9: 11-1: 0 methanol: gradient elution is carried out on the water mixed solution to obtain 2 components which are respectively named as a component Fr.1.1 and a component Fr.1.2 according to the order of peak appearance of the eluent; the component fr.1.1 may be isocratic eluted through normal phase silica gel column chromatography, and the isocratic elution may be performed using a volume ratio of 3: 1 petroleum ether: eluting with ethyl acetate mixture to obtain 3 components, and respectively naming the components as Fr.1.1.1, Fr.1.1.2 and Fr.1.1.3 according to the order of peak appearance of the eluate; the fraction fr.1.1.1 may be isocratic eluted through normal phase silica gel column chromatography, the isocratic elution d may be a volume ratio of 50: 1 dichloromethane: eluting with acetone mixed solution to obtain 4 components, and respectively naming the components as Fr.1.1.1.1, Fr.1.1.1.2, Fr.1.1.1.3 and Fr.1.1.1.4 according to the order of peak appearance of the eluent; the fraction fr.1.1.1.3 can be purified to give compound 9.
In some embodiments, a method of making compound 10, comprising: step S1: carrying out fermentation culture on monascus purpureus A2(Aspergillus puniceus A2) in a culture medium to obtain a fermentation product; then, the user can use the device to perform the operation,
step S2: extracting the fermentation product obtained in the step S1 to obtain a crude extract, and separating and purifying the crude extract to obtain a compound;
the separation and purification in step S2 may include: performing gradient elution a by normal phase silica gel column chromatography, wherein the gradient elution a is performed by using a volume ratio of 8-10 times of column volume of 1: 0-3: 1 petroleum ether: ethyl acetate mixture was subjected to gradient elution and detected using Thin Layer Chromatography (TLC); combining similar components to obtain 2 components, and respectively naming the components as a component Fr.1 and a component Fr.2 according to the order of peak appearance of eluent; the component fr.2 may be eluted through a reverse phase silica gel column gradient h, which may be a volume ratio using 5-8 column volumes of 3: 7-1: 0 methanol: gradient elution is carried out on the water mixed solution to obtain 3 components which are respectively named as a component Fr.2.1, a component Fr.2.2 and a component Fr.2.3 according to the order of peak appearance of the eluent; the component Fr.2.1 can be subjected to isocratic elution i through a sephadex column, and the isocratic elution i can be performed by using methanol with 6-9 times column volume to obtain 1 component which is named as the component Fr.2.1.1; the fraction fr.2.1.1 is purified to give compound 10.
In some embodiments, a method of making compound 11, comprising: step S1: carrying out fermentation culture on monascus purpureus A2(Aspergillus puniceus A2) in a culture medium to obtain a fermentation product; then, the user can use the device to perform the operation,
step S2: extracting the fermentation product obtained in the step S1 to obtain a crude extract, and separating and purifying the crude extract to obtain a compound;
the separation and purification in step S2 may include: performing gradient elution a by normal phase silica gel column chromatography, wherein the gradient elution a is performed by using a volume ratio of 8-10 times of column volume of 1: 0-3: 1 petroleum ether: ethyl acetate mixture was subjected to gradient elution and detected using Thin Layer Chromatography (TLC); combining similar components to obtain 2 components, and respectively naming the components as a component Fr.1 and a component Fr.2 according to the order of peak appearance of eluent; the fraction fr.2 may be eluted through a reverse phase silica gel column gradient h, which may be a volume ratio using 5-8 column volumes of 3: 7-1: 0 methanol: gradient elution is carried out on the water mixed solution to obtain 3 components which are respectively named as a component Fr.2.1, a component Fr.2.2 and a component Fr.2.3 according to the order of peak appearance of the eluent; the component Fr.2.1 can be subjected to isocratic elution i through a sephadex column, and the isocratic elution i can be performed by using methanol with 6-9 times column volume to obtain 1 component which is named as the component Fr.2.1.1; the fraction Fr.2.1.1 is purified to give compound 11.
In one embodiment, a method of making a compound, comprising:
step S1: carrying out fermentation culture on monascus purpureus A2 in a culture medium to obtain a fermentation product; then, the user can use the device to perform the operation,
step S2: extracting the fermentation product obtained in the step S1 to obtain a crude extract, and separating and purifying the crude extract to obtain a compound; the compound comprises at least one of the following compounds:
the separation and purification in the step S2 includes: performing gradient elution a by normal phase silica gel column chromatography, wherein the gradient elution a is performed by using a volume ratio of 8-10 times of column volume of 1: 0-3: 1 petroleum ether: ethyl acetate mixture was subjected to gradient elution and detected using Thin Layer Chromatography (TLC); combining similar components to obtain 2 components, and respectively naming the components as a component Fr.1 and a component Fr.2 according to the order of peak appearance of eluent;
and (b) performing gradient elution on the component Fr.1 through a reverse phase silica gel column, wherein the gradient elution b is performed by using a volume ratio of 5-8 times of column volume of 9: 11-1: 0 methanol: gradient elution is carried out on the water mixed solution to obtain 2 components which are respectively named as a component Fr.1.1 and a component Fr.1.2 according to the order of peak appearance of the eluent;
the component Fr.1.1 is subjected to isocratic elution c by normal phase silica gel column chromatography, wherein the isocratic elution c is a method for preparing the component Fr by using a volume ratio of 8-10 times of column volume to 3: 1 petroleum ether: eluting with ethyl acetate mixture to obtain 3 components, and respectively naming the components as Fr.1.1.1, Fr.1.1.2 and Fr.1.1.3 according to the order of peak appearance of the eluate;
the component Fr.1.1.1 is subjected to isocratic elution d by normal phase silica gel column chromatography, wherein the isocratic elution d is prepared by performing column chromatography on a normal phase silica gel column chromatography column, wherein the volume ratio of 10-15 times of column volume is 50: 1 dichloromethane: eluting with acetone mixed solution to obtain 4 components, and respectively naming the components as Fr.1.1.1.1, Fr.1.1.1.2, Fr.1.1.1.3 and Fr.1.1.1.4 according to the order of peak appearance of the eluent;
the component Fr.1.1.1.3 is purified to obtain a compound 9;
the component Fr.1.1.1.4 is purified to obtain a compound 8;
performing isocratic elution e on the component Fr.1.1.2 through normal-phase silica gel column chromatography, wherein the isocratic elution e is eluted by using dichloromethane with 10-15 times column volume to obtain 1 component which is named as a component Fr.1.1.2.1; purifying the component Fr.1.1.2.1 to obtain a compound 5 and a compound 6;
the component Fr.1.1.3 is subjected to isocratic elution f by normal phase silica gel column chromatography, wherein the isocratic elution f is eluted by using dichloromethane with 10-15 times column volume to obtain 2 components which are respectively named as a component Fr.1.1.3.1 and a component Fr.1.1.3.2 according to the order of peak appearance of eluent; purifying the component Fr.1.1.3.1 to obtain a compound 3 and a compound 4;
and (2) performing gradient elution on the component Fr.1.2 by normal-phase silica gel column chromatography, wherein the gradient elution is that the gradient elution g is performed by using a volume ratio of 8-10 times of column volume of 400: 1-1: 1 dichloromethane: gradient elution is carried out on the methanol mixed solution to obtain 2 components which are respectively named as a component Fr.1.2.1 and a component Fr.1.2.2 according to the order of peak appearance of the eluent; purifying the component Fr.1.2.2 to obtain a compound 7;
and (3) performing gradient elution h on the component Fr.2 through a reverse phase silica gel column, wherein the gradient elution h is a gradient elution method which uses a volume ratio of 5-8 times of column volume of 3: 7-1: 0 methanol: gradient elution is carried out on the water mixed solution to obtain 3 components which are respectively named as a component Fr.2.1, a component Fr.2.2 and a component Fr.2.3 according to the order of peak appearance of the eluent;
performing isocratic elution i on the component Fr.2.1 through a sephadex column, wherein the isocratic elution i is performed by using methanol with 6-9 times column volume to obtain 1 component which is named as a component Fr.2.1.1; the component Fr.2.1.1 is purified to obtain a compound 10 and a compound 11;
purifying the component Fr.2.2 to obtain a compound 2;
and (3) performing isocratic elution j on the component Fr.2.3 through a sephadex column, wherein the isocratic elution j is performed by using methanol with 6-9 times column volume to obtain a component Fr.2.3.1, and purifying the component Fr.2.3.1 to obtain the compound 1.
In a third aspect, the invention provides a compound which is Compound 1 or a pharmaceutically acceptable salt thereof or Compound 2 or a pharmaceutically acceptable salt thereof,
in a fourth aspect, the present invention provides a use of the above compound or a pharmaceutically acceptable salt thereof.
In some embodiments, the use of a compound, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for inhibiting nitric oxide release from a macrophage,
in some embodiments, the use of a compound, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating or ameliorating inflammation,
advantageous effects
Compared with the prior art, one embodiment of the invention has at least one of the following advantages:
(1) the monascus granatum A2(Aspergillus puniceus A2) provided by the invention is adopted for biosynthesis, so that various compounds with anti-inflammatory effects can be prepared, a new compound source is provided for development and treatment of inflammatory diseases, the method is simple to operate, and the obtained compounds are high in yield.
(2) The monascus granatum A2(Aspergillus punicius A2), the fermentation culture temperature and the fermentation culture time are adopted for biosynthesis, so that various compounds with anti-inflammatory effects can be prepared, a new compound source is provided for development and treatment of inflammatory diseases, the method is simple to operate, and the obtained compounds are high in yield.
(3) Compared with other culture mediums (such as potato glucose liquid culture medium), the culture medium provided by the invention is adopted for culture, which is beneficial to obtaining active substances with high yield and rich compound types.
(4) The separation and purification method provided by the invention is beneficial to obtaining various compounds with high purity.
Drawings
Fig. 1 is a statistical graph showing the effect of each compound on RAW264.7 cell viability in example 2 of the present invention.
FIG. 2 is a statistical graph showing the effect of each compound in example 2 of the present invention on NO release from RAW264.7 cells. Note: p < 0.0001; # # p <0.001 and # # p < 0.0001.
In the figure, 1,2, 3, 4, etc. on the abscissa represent compounds such as compound 1, compound 2, compound 3, compound 4, etc., "-" indicates no addition, "+" indicates addition, for example, LPS under compound 1 "+" indicates addition of LPS to compound 1 group.
Description of the terms
In the context of the present invention, all numbers disclosed herein are approximate values, regardless of whether the word "about" or "approximately" is used. Based on the numbers disclosed, the numerical values of each number may vary by less than + -10% or reasonably as recognized by one of ordinary skill in the art, such as by + -1%, + -2%, + -3%, + -4%, or + -5%.
The term "and/or" should be understood to mean any one of the options or a combination of any two or more of the options.
The term "HPLC" refers to high performance liquid chromatography.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention. The examples do not specify particular techniques or conditions, and are performed according to techniques or conditions described in literature in the art or according to the product specification. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
To illustrate the invention, the following examples are set forth. It is to be understood that the invention is not limited to these embodiments, but is provided as a means of practicing the invention.
LPS represents lipopolysaccharide; NO represents nitric oxide; mol represents mol; μ mol/L means micromoles per liter; m represents mol per liter; μ M means micromoles per liter; l represents liter; mg/mL means milligrams per milliliter. OD 548 The absorbance value at 548nm is indicated.
Example 1: preparation of 11 compounds
(1) 3.2-3.5mL of seed liquid culture medium of Monascus purpureus A2(Aspergillus puniceus A2) was added into a 1L flask containing the culture medium (wherein the culture medium in the 1L flask comprises 130g of rice, 5.6g of sea salt and 170mL of water), and the mixture was subjected to fermentation culture at 25 ℃ for 50 days, followed by 230 flasks co-fermentation culture. After fermentation, extracting with ethyl acetate, and concentrating the fermented crude extract under reduced pressure;
(2) combining the ethyl acetate extracting solution in the step (1), concentrating under reduced pressure to obtain a crude extract (305.3g), performing gradient elution by using normal phase silica gel column chromatography (100-200 meshes, petroleum ether-ethyl acetate volume ratio of 1: 0-3: 1), and detecting by using Thin Layer Chromatography (TLC); combining similar components to obtain 2 components, and respectively naming the components as a component Fr.1 and a component Fr.2 according to the order of peak appearance of eluent.
(3) And (3) carrying out gradient elution on the component Fr.1 in the step (2) through a reverse phase silica gel column (methanol-water, volume ratio of 9: 11-1: 0) to obtain 2 components, and respectively naming the components as a component Fr.1.1 and a component Fr.1.2 according to the order of peak appearance of eluent. The component Fr.1.1 is subjected to isocratic elution by normal phase silica gel column chromatography (200 meshes and 300 meshes, and the volume ratio of petroleum ether to ethyl acetate is 3: 1) to obtain 3 components which are respectively named as a component Fr.1.1.1, a component Fr.1.1.2 and a component Fr.1.1.3 according to the order of peak appearance of eluent. The component Fr.1.1.1 is subjected to isocratic elution by normal phase silica gel column chromatography (200-300 mesh, dichloromethane-acetone 50: 1) to obtain 4 components which are respectively named as a component Fr.1.1.1.1.1, a component Fr.1.1.2, a component Fr.1.1.3 and a component Fr.1.1.1.4 according to the order of peak appearance of eluent. The fraction Fr.1.1.1.3 was isolated and purified by semi-preparative HPLC [5C18-MS-II column (250 mm. times.10 mm, 5 μm) ] (acetonitrile-water volume ratio 11: 9, 2mL/min) to give compound 9(84.8mg) with a retention time of 30 min.
The fraction Fr.1.1.1.4 was isolated and purified by semi-preparative HPLC [5C18-MS-II column (250 mm. times.10 mm, 5 μm) ] (acetonitrile-water volume ratio 13: 7, 2mL/min) to give compound 8(29.1mg) with a retention time of 35 min.
The component Fr.1.1.2 is isocratic eluted by normal phase silica gel column chromatography (200-300 mesh, dichloromethane) to obtain 1 component which is named as component Fr.1.1.2.1. The fraction Fr.1.1.2.1 was separated and purified by semi-preparative HPLC [5C18-MS-II column (250 mm. times.10 mm, 5 μm) ] (acetonitrile-water volume ratio 7: 3, 2mL/min) to give compound 5(9.7mg) and compound 6(23.1mg) with retention times of 30min and 45min, respectively.
The component Fr.1.1.3 is isocratic eluted by normal phase silica gel column chromatography (200-300 mesh, dichloromethane) to obtain 2 components which are respectively named as a component Fr.1.1.3.1 and a component Fr.1.1.3.2 according to the order of peak appearance of eluent. The fraction Fr.1.1.3.1 was separated and purified by semi-preparative HPLC [5C18-MS-II column (250 mm. times.10 mm, 5 μm) ] (acetonitrile-water volume ratio 11: 9, 2mL/min) to give compound 3(16mg) and compound 4(24.2mg) with retention times of 25min and 40min, respectively.
The component Fr.1.2 is subjected to gradient elution by normal phase silica gel column chromatography (200 meshes and 300 meshes, dichloromethane-methanol 400: 1-1: 1) to obtain 2 components which are respectively named as a component Fr.1.2.1 and a component Fr.1.2.2 according to the order of peak appearance of eluent.
The fraction Fr.1.2.2 was isolated and purified by semi-preparative HPLC [5C18-MS-II column (250 mm. times.10 mm, 5 μm) ] (acetonitrile-water volume ratio 39: 11, 2mL/min) to give compound 7(19.5mg) with a retention time of 38 min.
(4) And (3) carrying out gradient elution on the component Fr.2 obtained in the step (2) through a reverse phase silica gel column (methanol-water, volume ratio of 3: 7-1: 0) to obtain 3 components, and respectively naming the component Fr.2.1, the component Fr.2.2 and the component Fr.2.3 according to the order of peak appearance of eluent.
The fraction fr.2.1 was isocratically eluted using a sephadex column (methanol) to give 1 fraction, designated fraction fr.2.1.1.
The fraction Fr.2.1.1 was separated and purified by semi-preparative HPLC [5C18-MS-II column (250 mm. times.10 mm, 5 μm) ] (acetonitrile-water volume ratio 1: 9, 2mL/min) to give compound 10(5.9mg) and compound 11(23.7mg) with retention times of 15min and 25min, respectively.
The fraction Fr.2.2 was isolated and purified by semi-preparative HPLC [5C18-MS-II column (250 mm. times.10 mm, 5 μm) ] (methanol-water volume ratio 1: 4, 2mL/min) to give compound 2(65mg, retention time 25 min).
The fraction Fr.2.3 was isocratically eluted using sephadex column (methanol) to give 1 fraction, designated fraction Fr.2.3.1, which fraction Fr.2.3.1 was isolated and purified by semi-preparative HPLC [5C18-MS-II column (250 mm. times.10 mm, 5 μm) ] (methanol-water volume ratio 4: 21, 2mL/min) to give compound 1(186mg, retention time 22 min).
(5) NMR and HRESIMS analyses were performed on each of the compounds obtained in steps (3) and (4).
Compound 1: deep red solid, according to high resolutionMass Spectrometry HRESIMS (M/z 237.0727, [ M + Na ]] + ) The compound is presumed to have a formula of C by combining NMR data 10 H 14 O 5 The unsaturation degree is 4. In that 1 H NMR Spectrum (600MHz, DMSO-d) 6 ) In, two methyl groups [ delta ] are indicated H 0.87(3H,d,J＝6.4Hz),3.62(3H,s)]Two olefinic hydrogens [ delta ] H 6.20(1H,s)]And two Ar-OH [ delta ] H 8.86(1H, s) and δ H 9.07(1H,s)]Is present. It is composed of 13 C NMR(600MHz,DMSO-d 6 ) Giving a 10 carbon signal comprising 2 methyl groups (. delta.) C 19.8,60.4),4 methines (. delta.) C 71.2,72.3,103.0,104.7) and 4 quaternary carbons (. delta.) C 137.1,138.3,150.3,153.5). The existence of asymmetric tetra-substituted benzene ring [ delta ] in the compound can be inferred by combining 1DNMR and HSQC spectral data H 6.20(H-4),6.20(H-6)；(δ C 102.9(C-4),104.6(C-6),137.2(C-1),138.3(C-2),150.4(C-3),153.6(C-5))]. Process for preparing compounds 1 H- 1 H COSY combined with HSQC spectrogram prompts a spin coupling system CH 3 -the presence of-CH-CH-. Combined with HMBC Spectroscopy, H-7 (. delta.) H 4.48) and C-6 (. delta.)) C 104.7) and C-2 (. delta.)) C 138.3) having correlation CH can be determined 3 the-CH-fragment being attached at the C-1 position, H 3 -O(δ H 3.62) and C-2 (. delta.)) C 138.3) remote correlation, determining that the methoxy group is attached at the C-2 position. Chemical shifts (delta) for 7-OH were determined from the COSY correlation between 7-OH and H-7 H 4.94, d, J ═ 3.4 Hz). The HMBC spectrum suggests that 3-OH is correlated with C-2/C-4, 5-OH is correlated with C-4/C-6, and it can be determined that the two phenolic hydroxyl groups are linked at the C-3 and C-5 positions. In conclusion, compound 1 was identified as 1- (2-methoxy-3, 5-dihydroxy) phenyl-1, 2-propanediol having a novel structure. Compound 1 structure and main 1 H- 1 The H COSY and HMBC related signals are as follows:
table 1: process for preparation of Compound 1 1 H (600MHz) and 13 c (600MHz) Nuclear magnetic data (DMSO-d) 6 )
Compound 2: yellow oil, according to high resolution Mass Spectrometry HRESIMS (M/z 235.0576, [ M + Na ]] + ) The compound is presumed to have the molecular formula C by combining NMR data 10 H 12 O 5 The unsaturation degree is 5. In that 1 H-NMR spectra (600MHz, DMSO-d) 6 ) Middle, delta H 6.12(1H, d, J ═ 2.9Hz) and δ H 6.27(1H, d, J ═ 2.9Hz) suggests only 2 meta-coupled aromatic proton signals, i.e. tetra-substitution on the aromatic ring, with donor group substitution that shifts the aromatic hydrogens to high field; delta H 9.03(1H, s) and δ H 9.27(1H, s) indicates the presence of 2 Ar-OH, δ H 2.03(3H, s) indicates the presence of-CO-CH 3 ，δ H 3.63(3H, s) indicates the presence of-O-CH 3 . From the coupling constant and COSY spectrum, delta can be known H 5.10(1H, d, J ═ 4.7Hz) and δ H 5.59(1H, d, J ═ 4.7Hz) coupled to each other and recombined 13 C-NMR(600MHz,DMSO-d 6 ) From the HSQC spectrum, δ H 5.59(1H, d, J ═ 4.7Hz) uncorrelated carbon signals are alcohol hydroxyl proton signals, and delta is the alcohol hydroxyl proton signal H 5.10(1H, d, J ═ 4.7Hz) and δ C 74.7, it is suggested that the presence of the-CHOH-fragment, combined with the HMBC spectrum, can be deduced as the fragment-CHOH-CO-CH 3 And is attached at the 1-position of the phenyl ring. Bonding of 13 C-NMR showed 6 aromatic carbon signals (. delta.) C 153.9,151.0,138.5,134.2,105.2,104.1),1 carbonyl carbon signal δ C 208.3, 1 methyl carbon Signal δ C 26.0 and 1 methoxy carbon signal delta C 60.6. From the HMBC spectrum, C-7 (. delta.) is known C 74.7) with aromatic protons H-6, C-7-OH, H 3 -9 remote correlation, H-7 (delta) H 5.10) and δ C 134.2，δ C 138.5，δ C 105.2，C-8(δ C 208.3) remote correlation, and methoxy proton delta H+ 3.63 with δ only C 138.5 remote correlation, it was thus determined that the ortho position of the 1-position of the phenyl ring is substituted by 1 methoxy group and 1 is not substituted by 1 methoxy group, respectivelySubstituted aromatic protons, with meta-substituted aromatic protons. In conclusion, compound 2 was identified as 1- (2-methoxy-3, 5-dihydroxy) phenyl-1-hydroxy-acetone with a novel structure. Compound 2 structure and main 1 H- 1 The H COSY and HMBC related signals are as follows:
table 2: process for preparation of Compound 2 1 H (600MHz) and 13 c (600MHz) Nuclear magnetic data (DMSO-d) 6 )
Compound 3:
a light yellow powder; molecular formula C 18 H 11 ClO 7 。 1 H NMR(600MHz,DMSO-d 6 ):δ H 13.38(s,1-OH),7.94(dd,J＝9.1,3.7Hz,H-6),7.41(dd,J＝9.1,4.0Hz,H-5),6.77(d,J＝2.8Hz,H-4'),6.64(d,J＝3.6Hz,,H-4),6.40(d,J＝10.4Hz,H-1'),5.62(d,J＝2.8Hz,H-3'),3.88(s,8-OCH 3 ). 13 C NMR(150MHz,DMSO-d 6 ):δ C 61.5(8-OCH 3 ,CH 3 ),88.4(C-4,CH),89.6(C-2',C),104.5(C-9a,C),106.7(C-3',CH),111.7(C-2,C),115.0(C-5,CH),115.6(C-8a,C),117.3(C-1',CH),123.3(C-7,C),136.0(C-6,CH),148.2(C-4',CH),154.9(C-8,C),155.5(C-10a,C),157.0(C-4a,C),158.1(C-1,C),165.7(C-3,C),180.1(C-9,C)。
Compound 4:
a light yellow powder; molecular formula C 20 H 15 ClO 7 。 1 H NMR(600MHz,CDCl 3 ):δ H 7.15(s,H-5),6.75(d,J＝7.0Hz,H-1'),6.73(s,H-4),6.47(s,H-4'),5.34(s,H-3'),4.74(d,J＝7.0,H-2'),4.03(s,1-OCH 3 ),3.95(s,6-/8-OCH 3 ). 13 C NMR(150MHz,CDCl 3 ):δ C 48.3(C-2',CH),56.8(6-OCH 3 ,CH 3 ),62.0(1-OCH 3 ,CH 3 ),62.1(8-OCH 3 ,CH 3 ),94.8(C-4,CH),103.3(C-3',CH),111.6(C-2,C),112.8(C-1',CH),116.0(C-5,CH),118.0(C-8a,C),118.5(C-9a,C),123.1(C-7,C),144.9(C-6,C),145.3(C-4'/4a,CH,C),148.2(C-10a,C),156.9(C-8,C),158.5(C-1,C),163.1(C-3,C),174.1(C-9,C)。
Compound 5:
a white powder; molecular formula C 20 H 15 ClO 7 。 1 H NMR(600MHz,DMSO-d 6 ):δ H 7.84(d,J＝9.1,H-6),7.41(d,J＝9.1,H-5),6.93(d,J＝6.9Hz,H-1'),6.76(t,J＝2.4Hz,H-4'),5.40(t,J＝2.6Hz,H-3'),4.86(dt,J＝6.9,2.2Hz,H-2'),3.92(s,4-OCH 3 ),3.89(s,1-OCH 3 ),3.87(s,8-OCH 3 ). 13 C NMR(150MHz,DMSO-d 6 ):δ C 47.7(C-2'.CH),60.9(4-OCH 3 ,CH 3 ),61.4(8-OCH 3 ,CH 3 ),61.6(1-OCH 3 ,CH 3 ),103.2(C-3',CH),111.5(C-9a,C),113.1(C-1',CH),114.7(C-5,CH),117.5(C-8a,C),118.7(C-2,C),122.8(C-7,C),127.5(C-4,C),134.6(C-6,CH),145.6(C-4',CH),150.0(C-4a,C),150.6(C-1,C),153.5(C-3,C),154.6(C-10a,C),154.8(C-8,C),172.9(C-9,C)。
Compound 6:
a light yellow powder; molecular formula C 19 H 13 ClO 6 。 1 H NMR(600MHz,DMSO-d 6 ):δ H 7.81(d,J＝9.0Hz,H-6),7.31(d,J＝9.0Hz,H-5),6.89(d,J＝6.9Hz,H-1'),6.81(s,H-4),6.74(t,J＝2.4Hz,H-4'),5.40(t,J＝2.5Hz,H-3'),4.85(dt,J＝7.0,2.5Hz,H-2'),3.94(s,1-OCH 3 ),3.87(s,8-OCH 3 ). 13 C NMR(150MHz,DMSO-d 6 ):δ C 47.5(C-2',CH),61.4(1-OCH 3 ,CH 3 ),61.4(8-OCH 3 ,CH 3 ),93.6(C-4,CH),103.3(C-3',CH),110.7(C-9a,C),112.7(C-1',CH),114.4(C-5,CH),117.6(C-2,C),117.8(C-8a,C),122.7(C-7,C),134.5(C-6,CH),145.4(C-4',CH),154.7(C-10a,C),154.8(C-8,C),156.2(C-1,C),157.8(C-4a,C),162.6(C-3,C),172.8(C-9,C)。
Compound 7:
a yellow powder; molecular formula C 22 H 18 O 7 。 1 H NMR(600MHz,DMSO-d 6 ):δ H 12.16(s,C1-OH),11.53(s,C8-OH),7.48(d,J＝8.3Hz,H-6),6.79(d,J＝2.8Hz,H-4'),6.71(d,J＝8.3Hz,H-7),6.62(s,H-4),6.44(s,C2'-OH),6.41(s,H-1'),5.63(d,J＝2.8Hz,H-3'),5.25(t,J＝6.8Hz,H-2”),3.39(d,J＝7.5Hz,H-1”),1.74(s,H-5”),1.68(s,H-4”). 13 C NMR(150MHz,DMSO-d 6 ):δ C 17.7(C-5”,CH 3 ),25.5(C-4”,CH 3 ),26.8(C-1”,CH 2 ),88.4(C-2',C),90.3(C-4,CH),102.9(C-9a,C),106.7(C-8a,C),106.7(C-3',CH),110.4(C-7,CH),111.9(C-2,C),117.3(C-1',CH),119.1(C-5,C),121.6(C-2”,CH),132.6(C-3”,C),137.3(C-6,CH),148.3(C-4',CH),152.6(C-10a,C),157.2(C-1,C),157.8(C-4a,C),158.3(C-8,C),166.3(C-3,C),184.5(C-9,C)。
Compound 8:
a light red powder; molecular formula C 15 H 20 O 2 。 1 H NMR(600MHz,Methanol-d 4 ):δ H 6.14(m,H-2),6.09(d,J＝1.6Hz,H-4/H-6),5.97(m,H-10/H-11),5.54(tt,J＝13.7,6.9Hz,H-9/H-12),2.51(m,H-7),2.30(q,J＝7.3Hz,H-8),2.02(q,J＝7.2Hz,H-13),1.39(h,J＝7.4Hz,H-14),0.90(t,J＝7.4Hz,H-15). 13 C NMR(150MHz,Methanol-d 4 ):δ C 14.0(C-15,CH 3 ),23.7(C-14,CH 2 ),35.4(C-13,CH 2 ),35.7(C-8,CH 2 ),37.1(C-7,CH 2 ),101.1(C-2,CH),107.9(C-4/C-6,CH),131.9(C-10,CH),132.1(C-11,CH),132.2(C-9,CH),133.1(C-12,CH),145.5(C-5,C),159.3(C-1/C-3,C)。
Compound 9:
a light yellow powder; molecular formula C 16 H 20 O 4 。 1 H NMR(600MHz,DMSO-d 6 ):δ H 6.17(d,J＝2.4Hz,H-3),6.16(d,J＝2.4Hz,H-5),5.96(m,H-10/H-11),5.55(m,H-9/H-12),2.85(t,J＝7.8Hz,H-7),2.25(q,J＝7.8Hz,H-8),1.98(m,H-13),1.34(m,H-14),0.84(t,J＝7.4Hz,H-15). 13 C NMR(150MHz,DMSO-d 6 ):δ C 13.6(C-16,C),22.2(C-15,CH 3 ),34.2(C-14,CH 2 ),34.4(C-8,CH 2 ),35.6(C-9,CH),100.9(C-3,C),104.9(C-1,C),110.3(C-5,C),130.5(C-12,CH),130.6(C-11,CH),131.5(C-13,CH 2 ),132.2(C-10,CH),146.6(C-6,C),161.9(C-2,CH),164.2(C-4,CH),172.9(C-7,CH 2 )。
Compound 10:
colorless oil; molecular formula C 9 H 14 O 3 。 1 H NMR(600MHz,DMSO-d 6 ):δ H 4.99(d,J＝4.2Hz,H-5),3.66(m,H-4a),3.34(d,J＝1.6Hz,H-4b),1.88(s,H-9),1.66(s,H-8),1.05(s,H-7). 13 C NMR(150MHz,DMSO-d 6 ):δ C 11.2(C-8,CH 3 ),18.3(C-7,CH 3 ),21.1(C-9,CH 3 ),38.8(C-4,CH 2 ),71.2(C-5,CH),75.9(C-6,C),127.2(C-2,C),152.0(C-3,C),201.1(C-1,C)。
Compound 11:
a white powder; molecular formula C 6 H 10 O 3 。 1 H NMR(600MHz,DMSO-d 6 ):δ H 4.37(td,J＝10.7,4.2Hz,H-6a),4.24(ddd,J＝10.9,5.4,4.0Hz,H-6b),2.49(m,H-5)1.84(m,H-3a),1.69(m,H-3b),1.20(s,H-7). 13 C NMR(150MHz,DMSO-d 6 ):δ C 29.3(C-7,CH 3 ),35.3(C-5,CH 2 ),44.5(C-3,CH 2 ),65.7(C-6,CH 2 ),66.8(C-4,C),170.6(C-2,C)。
Example 2: EXAMPLE 1 evaluation of anti-inflammatory Activity in vitro of 11 Compounds
(1) RAW264.7 cell culture and administration
Taking the para-RAW 264.7 cells in logarithmic growth phase at 2X 10 4 The density of each well was plated in 96-well plates (6 wells per group), and the cells were administered 24h after culture. The Control group (Control), the model group (1. mu.g/mL LPS stimulation), and the administration groups of the compounds 1 to 11 (1. mu.g/mL LPS stimulation + compounds at a concentration of 20. mu.M (Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, Compound 9, Compound 10, Compound 11, respectively)) were incubated in a carbon dioxide incubator at 37 ℃ for 24 hours, supernatants from the wells were collected, and the NO content was measured according to the following procedure (2).
(2) Determination of the NO content
Taking 1mmol/L nitrite (NaNO) in the kit 2 ) Standard solution, diluted to NO 2 - The concentrations were 3.125. mu. mol/L, 6.25. mu. mol/L, 12.5. mu. mol/L, 25. mu. mol/L, and 50. mu. mol/L, respectively.
According to the experimental dosage, equal volumes of the reagent A and the reagent B are mixed to prepare the Griess reagent. Reagent A: 1mg/mL of N-1-naphthylethylenediamine hydrochloride solution. And (3) reagent B: 10mg/mL of a sulfanilic acid solution.
Adding 75 mu L of nitrite standard solution or a sample to be tested into a 96-well plate, adding 10 mu L of Griess reagent and 65 mu L of deionized water into each well, oscillating the 96-well plate to fully and uniformly mix the solution in the wells, reacting for 30min at room temperature, and testing the absorbance of the reaction solution at 548 nm. By OD 548 And (5) performing regression on the series of concentrations of the standard sample to obtain a standard curve. The concentration of the sample to be measured is calculated by a standard curve method.
(3) Detecting the cell activity effect of the sample on RAW264.7 cells
After administration, 5% CO at 37 deg.C 2 Culturing for 1d in an incubator under the condition, removing supernatant, and adding the mixture according to the proportion of 1: CCK8 of 9: the DMEM medium is cultured for 2 hours in a 100-microliter mode, and then the absorbance value of the DMEM medium is measured at the wavelength of 450nm by using a microplate reader, and the cell activity is calculated.
(4) Result processing
The effect of the sample on the cell viability of RAW264.7 cells was calculated as follows.
Cell viability (%) × (model group/experimental group-blank group)/(control group-blank group) × 100%.
Blank-absorbance of wells with medium and CCK8 solution, no cells
Model group-absorbance of wells with cells, CCK8 solution and LPS
Experimental group-absorbance of wells with cells, CCK8 solution, LPS and drug
Control-absorbance of wells with cells, CCK8 solution
(5) Results of the experiment
As shown in fig. 1, the experimental results show that 11 compounds have no inhibitory effect on RAW264.7 cell proliferation, which indicates that the compounds have no toxic effect, and can ensure that the anti-inflammatory experiment is performed under the condition that the sample has no obvious influence on the growth of cells.
As shown in fig. 2, the measurement results of NO content showed that 11 compounds had significant anti-inflammatory activity.
While the methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention within the intended scope, spirit and scope of the invention. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included within the present invention.
Claims (10)
1. Use of Monascus purpureus solanacearum A2 for the preparation of at least one compound selected from the group consisting of,
2. a method of preparing a compound comprising:
step S1: carrying out fermentation culture on monascus purpureus A2 in a culture medium to obtain a fermentation product; then, the user can use the device to perform the operation,
step S2: and (4) extracting the fermentation product obtained in the step S1 to obtain a crude extract, and separating and purifying the crude extract to obtain a compound.
3. The method of claim 2, the compound comprising at least one of:
4. the method of claim 2, the culture medium comprising rice, sodium chloride, and water; and/or the rice accounts for 40.0-45.0 wt%, the sodium chloride accounts for 1.5-2.0 wt%, and the balance is water, calculated by the total mass of the culture medium.
5. The method of claim 2, wherein the fermentation culture is at a culture temperature of 25 ℃ to 28 ℃; and/or the culture time of the fermentation culture is 42 days to 55 days.
6. The method of claim 2, the extracting comprising extracting with ethyl acetate.
7. The method according to claim 2, wherein the separation and purification in the step S2 comprises: performing gradient elution a by normal phase silica gel column chromatography, wherein the gradient elution a is performed by using a volume ratio of 8-10 times of column volume of 1: 0-3: 1 petroleum ether: gradient eluting with ethyl acetate mixture, and detecting with thin layer chromatography; combining similar components to obtain 2 components, and respectively naming the components as a component Fr.1 and a component Fr.2 according to the order of peak appearance of eluent;
and/or
And (b) performing gradient elution on the component Fr.1 through a reverse phase silica gel column, wherein the gradient elution b is performed by using a volume ratio of 5-8 times of column volume of 9: 11-1: 0 methanol: gradient elution is carried out on the water mixed solution to obtain 2 components which are respectively named as a component Fr.1.1 and a component Fr.1.2 according to the order of peak appearance of the eluent;
the component Fr.1.1 is subjected to isocratic elution c by normal phase silica gel column chromatography, wherein the isocratic elution c is a method for preparing the component Fr by using a volume ratio of 8-10 times of column volume to 3: 1 petroleum ether: eluting with ethyl acetate mixture to obtain 3 components, and respectively naming the components as Fr.1.1.1, Fr.1.1.2 and Fr.1.1.3 according to the order of peak appearance of the eluate;
and/or
The component Fr.1.1.1 is subjected to isocratic elution d by normal phase silica gel column chromatography, wherein the isocratic elution d is prepared by performing column chromatography on a normal phase silica gel column chromatography column, wherein the volume ratio of 10-15 times of column volume is 50: 1 dichloromethane: eluting with acetone mixed solution to obtain 4 components, and respectively naming the components as Fr.1.1.1.1, Fr.1.1.1.2, Fr.1.1.1.3 and Fr.1.1.1.4 according to the order of peak appearance of the eluent;
the component Fr.1.1.1.3 is purified to obtain a compound 9;
and/or
The component Fr.1.1.1.4 is purified to obtain a compound 8;
and/or
Performing isocratic elution e on the component Fr.1.1.2 through normal-phase silica gel column chromatography, wherein the isocratic elution e is eluted by using dichloromethane with 10-15 times column volume to obtain 1 component which is named as a component Fr.1.1.2.1; purifying the component Fr.1.1.2.1 to obtain a compound 5 and a compound 6;
and/or
The component Fr.1.1.3 is subjected to isocratic elution f by normal phase silica gel column chromatography, wherein the isocratic elution f is eluted by using dichloromethane with 10-15 times column volume to obtain 2 components which are respectively named as a component Fr.1.1.3.1 and a component Fr.1.1.3.2 according to the order of peak appearance of eluent; purifying the component Fr.1.1.3.1 to obtain a compound 3 and a compound 4;
and/or
And (2) performing gradient elution on the component Fr.1.2 by normal-phase silica gel column chromatography, wherein the gradient elution is that the gradient elution g is performed by using a volume ratio of 8-10 times of column volume of 400: 1-1: 1 dichloromethane: gradient elution is carried out on the methanol mixed solution to obtain 2 components which are respectively named as a component Fr.1.2.1 and a component Fr.1.2.2 according to the order of peak appearance of the eluent; purifying the component Fr.1.2.2 to obtain a compound 7;
and/or
And (3) performing gradient elution h on the component Fr.2 through a reverse phase silica gel column, wherein the gradient elution h is a gradient elution method which uses a volume ratio of 5-8 times of column volume of 3: 7-1: 0 methanol: gradient elution is carried out on the water mixed solution to obtain 3 components which are respectively named as a component Fr.2.1, a component Fr.2.2 and a component Fr.2.3 according to the order of peak appearance of the eluent;
performing isocratic elution i on the component Fr.2.1 through a sephadex column, wherein the isocratic elution i is performed by using methanol with 6-9 times column volume to obtain 1 component which is named as a component Fr.2.1.1; the component Fr.2.1.1 is purified to obtain a compound 10 and a compound 11;
and/or
Purifying the component Fr.2.2 to obtain a compound 2;
and/or
And (3) performing isocratic elution j on the component Fr.2.3 through a sephadex column, wherein the isocratic elution j is performed by using methanol with 6-9 times column volume to obtain a component Fr.2.3.1, and purifying the component Fr.2.3.1 to obtain the compound 1.
8. A compound that is Compound 1 or a pharmaceutically acceptable salt thereof or Compound 2 or a pharmaceutically acceptable salt thereof,
9. the application of any one of the following compounds or pharmaceutically acceptable salts thereof in preparing a medicament for inhibiting the release of nitric oxide from macrophages,
10. the use of a compound, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or alleviation of inflammation,
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