CN111704546A - Oxygen-containing cyclohexane derivative and preparation method and application thereof - Google Patents
Oxygen-containing cyclohexane derivative and preparation method and application thereof Download PDFInfo
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- CN111704546A CN111704546A CN202010607849.0A CN202010607849A CN111704546A CN 111704546 A CN111704546 A CN 111704546A CN 202010607849 A CN202010607849 A CN 202010607849A CN 111704546 A CN111704546 A CN 111704546A
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- 229910052760 oxygen Inorganic materials 0.000 title claims abstract description 18
- 239000001301 oxygen Substances 0.000 title claims abstract description 18
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- 125000000113 cyclohexyl group Chemical class [H]C1([H])C([H])([H])C([H])([H])C([H])(*)C([H])([H])C1([H])[H] 0.000 title abstract description 6
- 239000003814 drug Substances 0.000 claims abstract description 21
- 210000002437 synoviocyte Anatomy 0.000 claims abstract description 20
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims abstract description 13
- 206010039073 rheumatoid arthritis Diseases 0.000 claims abstract description 13
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 11
- 239000001257 hydrogen Substances 0.000 claims abstract description 11
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 claims abstract description 8
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 7
- 230000035755 proliferation Effects 0.000 claims abstract description 5
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 27
- 150000001934 cyclohexanes Chemical class 0.000 claims description 21
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- 241000776601 Fissistigma oldhamii Species 0.000 claims description 13
- 239000000284 extract Substances 0.000 claims description 13
- 239000012046 mixed solvent Substances 0.000 claims description 10
- 239000003208 petroleum Substances 0.000 claims description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- 239000003480 eluent Substances 0.000 claims description 9
- 125000001424 substituent group Chemical group 0.000 claims description 9
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 7
- JHIVVAPYMSGYDF-UHFFFAOYSA-N cyclohexanone Chemical class O=C1CCCCC1 JHIVVAPYMSGYDF-UHFFFAOYSA-N 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- 239000002808 molecular sieve Substances 0.000 claims description 7
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 6
- 238000004809 thin layer chromatography Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 238000004587 chromatography analysis Methods 0.000 claims description 5
- 238000004440 column chromatography Methods 0.000 claims description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- 239000002024 ethyl acetate extract Substances 0.000 claims description 3
- 150000002431 hydrogen Chemical class 0.000 claims description 3
- 239000007791 liquid phase Substances 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- 238000011068 loading method Methods 0.000 claims description 2
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical group CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 238000002791 soaking Methods 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 3
- 238000004128 high performance liquid chromatography Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 18
- 150000001875 compounds Chemical class 0.000 abstract description 7
- 230000003356 anti-rheumatic effect Effects 0.000 abstract description 3
- 125000004435 hydrogen atom Chemical class [H]* 0.000 abstract 1
- 229940079593 drug Drugs 0.000 description 13
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 11
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 11
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 6
- 241001189830 Fissistigma Species 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 229960000485 methotrexate Drugs 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 4
- 230000003833 cell viability Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 238000001647 drug administration Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- -1 benzopyran compound Chemical class 0.000 description 2
- 229940125904 compound 1 Drugs 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000010898 silica gel chromatography Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- XILIYVSXLSWUAI-UHFFFAOYSA-N 2-(diethylamino)ethyl n'-phenylcarbamimidothioate;dihydrobromide Chemical compound Br.Br.CCN(CC)CCSC(N)=NC1=CC=CC=C1 XILIYVSXLSWUAI-UHFFFAOYSA-N 0.000 description 1
- 241001081440 Annonaceae Species 0.000 description 1
- 235000009025 Carya illinoensis Nutrition 0.000 description 1
- 241001453450 Carya illinoinensis Species 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical compound C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 description 1
- 230000001174 ascending effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- VILAVOFMIJHSJA-UHFFFAOYSA-N dicarbon monoxide Chemical compound [C]=C=O VILAVOFMIJHSJA-UHFFFAOYSA-N 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 description 1
- UREBWPXBXRYXRJ-UHFFFAOYSA-N ethyl acetate;methanol Chemical compound OC.CCOC(C)=O UREBWPXBXRYXRJ-UHFFFAOYSA-N 0.000 description 1
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 1
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 125000004430 oxygen atom Chemical group O* 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 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
- 238000007619 statistical method Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
- C07C69/76—Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring
- C07C69/78—Benzoic acid esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- 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]
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- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
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- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/56—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
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- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
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- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2601/00—Systems containing only non-condensed rings
- C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
- C07C2601/16—Systems containing only non-condensed rings with a six-membered ring the ring being unsaturated
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- Animal Behavior & Ethology (AREA)
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- Rheumatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
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Abstract
The invention discloses an oxygen-containing cyclohexane derivative, the structural formula of which is as follows,
wherein R is1、R2、R3、R4、R5Respectively one of hydrogen, benzoyl and ethyl.The invention also discloses a preparation method of the optimized compound and application of the optimized compound in preparing a medicament for resisting rheumatoid arthritis. The invention discovers the activity of the oxygen-containing cyclohexane derivative in inhibiting the proliferation of synovial cells of rheumatoid arthritis for the first time, and the oxygen-containing cyclohexane derivative can be used for preparing the anti-rheumatoid arthritis medicament.
Description
Technical Field
The invention relates to the technical field of phytochemistry, in particular to an oxygen-containing cyclohexane derivative and a preparation method and application thereof.
Background
Fissistigma Oldhamii (Fissistigma titentangense Tsiang et al T.Li) is a plant of Fissistigma Oldhamii of Annonaceae. The plant can be used as medicine, and its stem and leaf can be used for treating fracture and edema, and its whole plant can be used for treating rheumatalgia, deadlimb, etc. The fissistigma angustifolia contains a large amount of compounds, and a new-structure compound with obvious drug effect needs to be extracted urgently, so that the medicinal value of the fissistigma angustifolia is fully exerted.
Disclosure of Invention
The invention aims to provide an oxygen-containing cyclohexane derivative which is extracted and separated from fissistigma angustifolia and has a new structure, has the activity of inhibiting the proliferation of synovial cells of rheumatoid arthritis, and can be applied to the preparation of drugs for resisting rheumatoid arthritis.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
an oxygen-containing cyclohexane derivative, which has the following structural formula:
wherein R is1、R2、R3、R4、R5Each is hydrogen, benzoyl or ethyl, namely each is independently selected from-H or-Bz or-Et.
Further, said R1、R2、R3、R4、R5Wherein 1 substituent is ethyl, 1 substituent is benzoyl and 3 substituents are hydrogen.
The above-mentioned oxygenated cyclohexane derivative is preferably represented by the following structural formula:
the above preferred oxocyclohexane derivative can be prepared by a process comprising the steps of:
(1) preparation of Fissistigma Oldhamii extract
Cold soaking or hot extracting dry stem of Fissistigma Oldhamii with ethanol solution to obtain extractive solution, and concentrating under reduced pressure to obtain Fissistigma Oldhamii extract;
(2) separating and purifying
Diluting the Fissistigma Oldhamii extract with water to obtain suspension, sequentially extracting with petroleum ether and ethyl acetate, concentrating the ethyl acetate extract to obtain extract, and separating by column chromatography, thin layer chromatography and molecular sieve chromatography to obtain the compound named fisigense B.
Further, in the step (1), the vacuum concentration is carried out at the temperature of 30-70 ℃ and the pressure of-0.06 to-0.15 MPa; the preferred temperature is 40-55 ℃, and the preferred pressure is-0.09 to-0.1 MPa; more preferably, the temperature is 45 ℃ and the pressure is-0.095 MPa.
Further, in the step (1), the volume concentration of the ethanol solution is 30-95%, preferably 70-80%, and more preferably 75%.
Further, in the step (2), the column chromatography conditions are as follows: and (3) loading the mixture to a 200-300-mesh silica gel column, wherein an ethyl acetate-petroleum ether mixed solvent with the ethyl acetate volume percentage of 30% is used as an eluent.
Further, in the step (2), the thin layer chromatography conditions are as follows: the developing solvent is ethyl acetate-petroleum ether mixed solvent with 50 percent of ethyl acetate volume percentage.
Further, in the step (2), the molecular sieve chromatography conditions are as follows: the molecular sieve is SephadexLH-20, and chloroform-methanol mixed solvent with chloroform volume percentage of 50% is used as eluent.
Further, in the step (2), the eluent of the high-performance liquid phase is acetonitrile and water with a volume ratio of 45: 55.
The application of the oxygen-containing cyclohexane derivative in preparing the anti-rheumatoid arthritis medicine is disclosed.
The application of the oxygen-containing cyclohexane derivative in preparing the medicine for inhibiting the proliferation of rheumatoid arthritis synovial cells is disclosed. Wherein, the synoviocytes are primary rat synoviocytes.
Compared with the prior art, the invention has the beneficial effects that: the invention extracts and separates a new structural compound-oxygen-containing cyclohexane derivative from fissistigma angustifolia, and the invention discovers the activity of the oxygen-containing cyclohexane derivative in inhibiting the proliferation of rheumatoid arthritis synovial cells for the first time, and can be used for preparing the anti-rheumatoid arthritis medicament.
Drawings
FIG. 1 is a hydrogen spectrum of a benzopyran compound fisigense B of example 1 of the present invention.
FIG. 2 is a carbon spectrum of a benzopyran compound fisigense B according to example 1 of the present invention.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.
An oxygen-containing cyclohexane derivative, which has the following structural formula:
wherein R is1、R2、R3、R4、R5Each is hydrogen, benzoyl or ethyl, namely each is independently selected from-H or-Bz or-Et. Preferably, said R is1、R2、R3、R4、R5Wherein 1 substituent is ethyl, 1 substituent is benzoyl and 3 substituents are hydrogen. The compounds of the above structure can be obtained by organic synthesis. The invention relates to aExtracting and separating the oxygenated cyclohexane derivative with the following structure from the Fissistigma Oldhamii:
example 1
The preparation of the oxygenated cyclohexane derivative comprises the following steps:
(1) extracting 10Kg dry stem of Fissistigma Oldhamii with 75% v/v ethanol solution for 3 times and 7 days to obtain extractive solution, concentrating under reduced pressure to obtain paste, and concentrating at 45 deg.C under-0.095 MPa to obtain 1000g Fissistigma Oldhamii extract.
(2) Diluting the extract with distilled water (3L) to obtain suspension, sequentially extracting with petroleum ether (3L × 3 times) and ethyl acetate (3L × 3 times), and concentrating the ethyl acetate extract to obtain extract (about 256g) at 45 deg.C under-0.095 MPa. Subjecting the extract to silica gel column chromatography, subjecting to silica gel column chromatography with petroleum ether-ethyl acetate mixed solvent (100: 0-0: 100, V/V) and ethyl acetate-methanol (100: 0-0: 100, V/V) in ascending polarity, and collecting fractions of about 400mL each time. Similar fractions were pooled and split into 9 fractions, Fr.1-9, by TLC detection.
(3) And Fr.3, eluting by using 200-300-mesh silica gel column, using an ethyl acetate-petroleum ether mixed solvent with ethyl acetate volume percentage of 30% as an eluent, performing thin layer chromatography by using an ethyl acetate-petroleum ether mixed solvent with ethyl acetate volume percentage of 50% as a developing agent, and merging fractions according to the chromatography effect to obtain 3 components Fr.3-1, Fr.3-2 and Fr.3-3.
(4) Fr.3-1 uses chloroform-methanol mixed solvent with 50% chloroform volume percentage as eluent to carry out molecular sieve Sephadex LH-20 column chromatography to remove pigment to obtain a component Fr.3-1-1, Fr.3-1-1 is analyzed and prepared by high performance liquid phase, the eluent is acetonitrile and water with the volume ratio of 45:55, and oxygen-containing cyclohexane derivative fisigense B is obtained. The hydrogen spectrum and carbon spectrum are shown in figure 1-2.
The structural identification result of the oxygen-containing cyclohexane derivative fisigense B is as follows:
fissigense B: is white powder with optical rotation of
HR-ESI-MS m/z 309.1333[M+H]+Is combined with1H-NMR and13c NMR indicates that the molecular formula is C16H20O6. According to1H,13The compound is an oxygenated cyclohexane derivative by C-NMR and DEPT1H-NMR and13c NMR data. In the HMBC spectrum, the correlation between-Et hydrogen and the carbon at the 6-position of cyclohexane shows that-Et is connected to the C-6 position through an oxygen atom; the hydrogen at the 3-position of cyclohexane is associated with the carbonyl carbon in the benzoyl group, indicating that the missing-Bz is at the 7-position. The structure of compound 1 was confirmed as shown in the above figure.
TABLE 1 preparation of Compound 11H-NMR and13C-NMR data
Example 2
This example differs from example 1 in that, in step (1), the ethanol solution has a volume concentration of 30%; in the step (1) and the step (2), the concentration temperature is about 35 ℃, and the pressure is-0.15 MPa. The product structure obtained was the same as in example 1.
Example 3
This example differs from example 1 in that, in step (1), the ethanol solution has a volume concentration of 95%; in the step (1) and the step (2), the concentration temperature is about 65 ℃, and the pressure is-0.06 MPa. The product structure obtained was the same as in example 1.
Test example: pharmacological Activity test
Experimental Material
Cell: synoviocytes (primary rat synoviocytes).
Cell culture solution: primary rat synovial cell culture medium.
Reagent: thiazole blue (3- (4, 5-dimethylthiozol-2-yl) -2, 5-diphenyltetrazolium bromide, MTT, Sigma). Lactate Dehydrogenase (LDH) kit (available from pecan). Methotrexate (MTX, Shanghai Processingequiz, Inc., Lot 20140307).
1.4 Instrument: 96-well cell culture plates; an infinite 200Pro multifunctional microplate reader.
Experimental methods
(1) MTT method for determining inhibitory effect of drug on synovial cells
Synovial cells were inoculated into 96-well plates (1 × 10) at log phase4Individual cells/well), set as blank group (no drug administration), methotrexate group (1 μ g/mL methotrexate administration), administration group (10, 50, 100 μ g/mL drug administration — fisssigene B prepared in example 1), and incubated overnight for experiment. After 48 hours of incubation in which cells were administered with different concentrations of drugs, 10. mu.L of MTT at a concentration of 5mg/mL was added to each well, and after further incubation for 4 hours, the culture supernatant was carefully discarded, 100. mu.L of DMSO was added to each well to dissolve formazan crystals, and after complete dissolution, the optical density (OD value) was measured at 570nm with a microplate reader after shaking 5min at room temperature. Cell viability was calculated from the OD values.
(2) LDH Activity assay to determine the Effect of drugs on synovial cell viability
Synovial cells were inoculated into 96-well plates (1 × 10) at log phase4Individual cells/well), set as a blank group (no drug administration), a methotrexate group (1. mu.g/mL methotrexate administration), and an administration group (10, 50, 100. mu.g/mL drug administration-fisssigene B prepared in example 1), incubated overnight in culture, and after the completion of cell culture in each group, cell supernatants were collected and LDH activity was detected by an LDH detection kit. The activity of LDH was calculated according to the following formula.
Statistical method
Experimental data were analyzed using SPSS 22.0 statistical software. Results are given as mean. + -. standard deviation
Show that differences between groups were analyzed by one-way ANOVA. With P<0.05 was considered statistically different.
Results of the experiment
(1) MTT method for determining inhibitory effect of drug on synovial cells
The results of MTT method for detecting cell proliferation after 48h of culture with different concentrations of drug are shown in Table 2 below. The result shows that the fisssigente A has good inhibition effect on synovial cells.
TABLE 2 synovial cell inhibition by each drug
Note: in comparison with the blank set, the results,*P﹤0.05，**P﹤0.01。
(2) LDH Activity assay to determine the Effect of drugs on synovial cell viability
The results of the LDH activity assay for cell viability after 48h incubation with different concentrations of drug are shown in Table 3 below. The results show that LDH activity (P <0.05 or P < 0.01) was increased in cell culture fluid after treatment with different concentrations of drug.
TABLE 3 Effect of each drug on synovial cell LDH Activity
Note: in comparison with the blank set, the results,*P﹤0.05，**P﹤0.01。
the results show that the oxygen-containing cyclohexane derivative fisigense B has a good inhibition effect on synovial cells and can improve the LDH activity in a cell culture solution. Experiments show that the oxygen-containing cyclohexane derivative has good effect of resisting rheumatoid arthritis and can be used for preparing medicines for resisting rheumatoid arthritis.
Claims (10)
1. An oxygen-containing cyclohexane derivative, which has the following structural formula:
wherein R is1、R2、R3、R4、R5Respectively one of hydrogen, benzoyl and ethyl.
2. The oxocyclohexane derivative as claimed in claim 1, wherein R is the same as R1、R2、R3、R4、R5Wherein 1 substituent is ethyl, 1 substituent is benzoyl and 3 substituents are hydrogen.
3. The oxocyclohexane derivative as set forth in claim 2, having a structural formula as follows:
4. a process for producing an oxygenated cyclohexane derivative according to claim 3, which comprises the steps of:
(1) preparation of Fissistigma Oldhamii extract
Cold soaking or hot extracting dry stem of Fissistigma Oldhamii with ethanol solution to obtain extractive solution, and concentrating under reduced pressure to obtain Fissistigma Oldhamii extract;
(2) separating and purifying
Diluting the Fissistigma Oldhamii extract with water to obtain suspension, sequentially extracting with petroleum ether and ethyl acetate, concentrating the ethyl acetate extract to obtain extract, and performing column chromatography, thin layer chromatography, molecular sieve chromatography, and high performance liquid chromatography to obtain the target product.
5. The method for producing an oxygenated cyclohexane derivative according to claim 4, wherein the ethanol solution in the step (1) has a volume concentration of 30 to 95%.
6. The method for producing an oxocyclohexane derivative according to claim 5, wherein the concentration in the steps (1) and (2) is carried out at a temperature of 30 to 70 ℃ and a pressure of-0.06 to-0.15 MPa.
7. The method for preparing an oxocyclohexane derivative according to any one of claims 4 to 6, wherein the column chromatography in the step (2) is performed under the following conditions: loading the mixture to a 200-300-mesh silica gel column, and taking an ethyl acetate-petroleum ether mixed solvent with the ethyl acetate volume percentage of 30% as an eluent; the thin layer chromatography conditions are as follows: the developing solvent is ethyl acetate-petroleum ether mixed solvent with 50 percent of ethyl acetate volume percentage.
8. The method for preparing an oxocyclohexane derivative according to claim 7, wherein the molecular sieve chromatography conditions in step (2) are: the molecular sieve is Sephadex LH-20, and chloroform-methanol mixed solvent with 50% chloroform volume percentage is used as eluent; the eluent of the high-efficiency liquid phase is acetonitrile and water with the volume ratio of 45: 55.
9. Use of the oxocyclohexane derivative as set forth in any one of claims 1 to 3 for the preparation of a medicament for treating rheumatoid arthritis.
10. Use of an oxygenated cyclohexane derivative according to any one of claims 1 to 3 for the manufacture of a medicament for inhibiting the proliferation of synovial cells of rheumatoid arthritis.
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