CN116675602B - Pinane diterpene and preparation method and application thereof - Google Patents
Pinane diterpene and preparation method and application thereof Download PDFInfo
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- 229930004069 diterpene Natural products 0.000 title claims abstract description 11
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- -1 Pinane diterpene Chemical class 0.000 title claims abstract description 9
- 229930006728 pinane Natural products 0.000 title abstract description 6
- XOKSLPVRUOBDEW-UHFFFAOYSA-N pinane of uncertain configuration Natural products CC1CCC2C(C)(C)C1C2 XOKSLPVRUOBDEW-UHFFFAOYSA-N 0.000 title abstract description 6
- 150000001875 compounds Chemical class 0.000 claims abstract description 37
- 210000004027 cell Anatomy 0.000 claims abstract description 23
- 238000000605 extraction Methods 0.000 claims abstract description 15
- 230000003110 anti-inflammatory effect Effects 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 13
- 238000000926 separation method Methods 0.000 claims abstract description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 37
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 24
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 13
- 239000000741 silica gel Substances 0.000 claims description 13
- 229910002027 silica gel Inorganic materials 0.000 claims description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 11
- 239000000126 substance Substances 0.000 claims description 10
- 239000000287 crude extract Substances 0.000 claims description 9
- 238000010828 elution Methods 0.000 claims description 9
- 239000000284 extract Substances 0.000 claims description 9
- 239000003208 petroleum Substances 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 8
- 230000002829 reductive effect Effects 0.000 claims description 7
- 239000011347 resin Substances 0.000 claims description 6
- 229920005989 resin Polymers 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- HWJHWSBFPPPIPD-UHFFFAOYSA-N ethoxyethane;propan-2-one Chemical compound CC(C)=O.CCOCC HWJHWSBFPPPIPD-UHFFFAOYSA-N 0.000 claims description 5
- 238000004811 liquid chromatography Methods 0.000 claims description 5
- 238000010992 reflux Methods 0.000 claims description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 4
- 238000011161 development Methods 0.000 claims description 4
- 210000003918 fraction a Anatomy 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 238000010025 steaming Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 150000004141 diterpene derivatives Chemical class 0.000 claims description 3
- ROAYSRAUMPWBQX-UHFFFAOYSA-N ethanol;sulfuric acid Chemical compound CCO.OS(O)(=O)=O ROAYSRAUMPWBQX-UHFFFAOYSA-N 0.000 claims description 3
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000000178 monomer Substances 0.000 claims description 3
- 238000010298 pulverizing process Methods 0.000 claims description 3
- 238000004809 thin layer chromatography Methods 0.000 claims description 3
- 101000610640 Homo sapiens U4/U6 small nuclear ribonucleoprotein Prp3 Proteins 0.000 claims description 2
- 101001110823 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 60S ribosomal protein L6-A Proteins 0.000 claims description 2
- 101000712176 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 60S ribosomal protein L6-B Proteins 0.000 claims description 2
- 102100040374 U4/U6 small nuclear ribonucleoprotein Prp3 Human genes 0.000 claims description 2
- 239000002260 anti-inflammatory agent Substances 0.000 claims description 2
- 230000008685 targeting Effects 0.000 claims description 2
- 238000005084 2D-nuclear magnetic resonance Methods 0.000 claims 1
- 235000008331 Pinus X rigitaeda Nutrition 0.000 claims 1
- 235000011613 Pinus brutia Nutrition 0.000 claims 1
- 241000018646 Pinus brutia Species 0.000 claims 1
- 229940121363 anti-inflammatory agent Drugs 0.000 claims 1
- 238000004896 high resolution mass spectrometry Methods 0.000 claims 1
- 230000002452 interceptive effect Effects 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 claims 1
- 238000000238 one-dimensional nuclear magnetic resonance spectroscopy Methods 0.000 claims 1
- 238000002390 rotary evaporation Methods 0.000 claims 1
- 239000002158 endotoxin Substances 0.000 abstract description 18
- 229920006008 lipopolysaccharide Polymers 0.000 abstract description 18
- 238000005481 NMR spectroscopy Methods 0.000 abstract description 11
- 239000000463 material Substances 0.000 abstract description 9
- 239000003814 drug Substances 0.000 abstract description 6
- 229940124599 anti-inflammatory drug Drugs 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 3
- 238000012827 research and development Methods 0.000 abstract description 3
- 238000012360 testing method Methods 0.000 abstract description 3
- 238000001819 mass spectrum Methods 0.000 abstract description 2
- 229930014626 natural product Natural products 0.000 abstract description 2
- 238000004440 column chromatography Methods 0.000 abstract 1
- 230000002757 inflammatory effect Effects 0.000 abstract 1
- 210000002540 macrophage Anatomy 0.000 abstract 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 6
- 229940079593 drug Drugs 0.000 description 5
- 238000010438 heat treatment Methods 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- QJMCKEPOKRERLN-UHFFFAOYSA-N N-3,4-tridhydroxybenzamide Chemical compound ONC(=O)C1=CC=C(O)C(O)=C1 QJMCKEPOKRERLN-UHFFFAOYSA-N 0.000 description 3
- 230000003698 anagen phase Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000003833 cell viability Effects 0.000 description 3
- 238000005100 correlation spectroscopy Methods 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 210000000988 bone and bone Anatomy 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 2
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- NEAQRZUHTPSBBM-UHFFFAOYSA-N 2-hydroxy-3,3-dimethyl-7-nitro-4h-isoquinolin-1-one Chemical compound C1=C([N+]([O-])=O)C=C2C(=O)N(O)C(C)(C)CC2=C1 NEAQRZUHTPSBBM-UHFFFAOYSA-N 0.000 description 1
- 208000006820 Arthralgia Diseases 0.000 description 1
- 241000092728 Eleutherococcus gracilistylus Species 0.000 description 1
- 206010021118 Hypotonia Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 238000000134 MTT assay Methods 0.000 description 1
- 231100000002 MTT assay Toxicity 0.000 description 1
- 241000208125 Nicotiana Species 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 208000004880 Polyuria Diseases 0.000 description 1
- 208000025747 Rheumatic disease Diseases 0.000 description 1
- 241000555745 Sciuridae Species 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 206010047601 Vitamin B1 deficiency Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 208000002894 beriberi Diseases 0.000 description 1
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000035619 diuresis Effects 0.000 description 1
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 description 1
- 208000017561 flaccidity Diseases 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 1
- 238000005570 heteronuclear single quantum coherence Methods 0.000 description 1
- 238000000990 heteronuclear single quantum coherence spectrum Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000007721 medicinal effect Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000009965 odorless effect Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 229930015704 phenylpropanoid Natural products 0.000 description 1
- 125000001474 phenylpropanoid group Chemical group 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000000552 rheumatic effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- FDDDEECHVMSUSB-UHFFFAOYSA-N sulfanilamide Chemical compound NC1=CC=C(S(N)(=O)=O)C=C1 FDDDEECHVMSUSB-UHFFFAOYSA-N 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C62/00—Compounds having carboxyl groups bound to carbon atoms of rings other than six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C62/30—Unsaturated compounds
- C07C62/32—Unsaturated compounds containing hydroxy or O-metal groups
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C2603/00—Systems containing at least three condensed rings
- C07C2603/02—Ortho- or ortho- and peri-condensed systems
- C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
- C07C2603/22—Ortho- or ortho- and peri-condensed systems containing three rings containing only six-membered rings
- C07C2603/26—Phenanthrenes; Hydrogenated phenanthrenes
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the technical field of natural product preparation, and in particular relates to a pinane diterpene and a preparation method and application thereof, wherein the new pinane diterpene is obtained by separating cortex acanthopanacis serving as a traditional Chinese medicine material through a combined column chromatography technology: 14-hydroxy-16-de-ethenyl-enantiomer-pimpinene-8, 11, 13-triene-17-carboxylic acid, the structure of which is determined by nuclear magnetic resonance, mass spectrum and other techniques. The Griess method is adopted to test a lipopolysaccharide-induced mouse mononuclear macrophage RAW264.7 cell release inflammatory factor model, and the anti-inflammatory activity of the compound is evaluated, so that the compound has certain anti-inflammatory activity. The invention obtains a new component, and defines the extraction and separation and structure identification method of the new component, the method is simple and convenient, and the component can be obtained repeatedly and continuously. Meanwhile, a material basis is provided for the research and development of new anti-inflammatory drugs.
Description
Technical Field
The invention belongs to the technical field of natural product preparation, and particularly relates to a new chemical structure of a sea squirrel diterpene extracted from cortex acanthopanacis and application thereof.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Cortex Acanthopancis of Acanthopanax gracilistylus of Acanthopanax of AraliaceaeAcanthopanax gracilistylusW.W.Smith) is mainly distributed in Hubei and He of ChinaThe Chinese medicinal materials are traditional Chinese medicinal materials commonly used in clinic in the provinces of south, shaanxi, anhui, zhejiang, liaoning, hebei and the like. The cortex acanthopanacis is originally carried in the book meridian, has the main effects of dispelling wind and removing dampness, tonifying liver and kidney, strengthening tendons and bones, inducing diuresis and relieving swelling, and is clinically mainly used for treating diseases such as rheumatic arthralgia, flaccidity of bones and muscles, infantile slow running, body deficiency and general power, edema, beriberi and the like, and the chemical components of the cortex acanthopanacis mainly comprise terpenes, sterols, organic acids, phenylpropanoids, flavonoids and the like. Modern pharmacology shows that cortex acanthopanacis has good anti-inflammatory activity.
In the prior art, the research on chemical components and medicinal effects of cortex acanthopanacis is less, and the plant cannot be reasonably and effectively utilized.
Disclosure of Invention
Based on the research background, the invention extracts the new-structure pimarane diterpenoid compound from the cortex acanthopanacis, performs in-vitro anti-inflammatory activity, and provides a material basis for the research and development of anti-inflammatory new drugs.
The invention discloses a kind of sea-pinane diterpene, the structural formula is shown in formula I,
。
the identification of the chemical components of formula I is referred to in the corresponding parts of the specific embodiments.
The invention discloses a preparation method of a pinane diterpene with a structural formula shown in formula I, which comprises the following steps:
(1) Preparing a crude extract;
(2) Carrying out fractional extraction;
(3) Separating and purifying;
(4) Identification of chemical components.
The method comprises the following steps:
(1) Pulverizing cortex Acanthopanacis, extracting with 70% ethanol under reflux, collecting middle and small polar parts, and steaming at 60deg.C to obtain crude extract;
(2) Dispersing the crude extract with warm water, extracting with diethyl ether, standing to completely layer, separating the extraction solvent, repeatedly extracting, mixing the extractive solutions, and concentrating under reduced pressure to obtain diethyl ether layer extract;
(3) Separating the ether layer extract by using an MCI resin column, performing gradient elution by using a methanol aqueous solution, wherein the fraction A passes through a forward silica gel chromatographic column, performing gradient elution by using a petroleum ether-acetone system, detecting by using a thin-layer silica gel plate, combining the same components, targeting to find spots with dark spots and fluorescence under an ultraviolet lamp in the silica gel thin-layer chromatography, and separating the component A1 with the target spots by using a liquid chromatography to obtain a target compound;
(4) The chemical structure of the target compound is determined by adopting high-resolution mass spectrum, 1D nuclear magnetic resonance spectrum and other technologies.
Further, in the step (1), the extraction mode of heating and refluxing is adopted for 3 times, 1 time h each time, so that the material extraction is ensured to be complete.
Further, in the step (1), the reduced pressure rotary steaming temperature is 57 ℃, so that the target component is prevented from being damaged by long-time high-temperature heating.
Further, in the step (2), the extraction is repeated for 3 times, and the volume ratio of diethyl ether to water layer used in the extraction is 1:1.
In the step (3), the MCI resin column is subjected to gradient elution by 40% -100% of methanol aqueous solution, so that the target component is separated from other interference components.
Further, in the step (3), the fraction A is subjected to forward silica gel chromatographic column and is subjected to gradient elution by petroleum ether-acetone system (100:1-1:1), so that the target component is further separated from other interference components.
Further, in the step (3), the observation wavelength of the dark spots is 254.+ -.2 nm, the observation wavelength of the fluorescence is 365.+ -.2 nm, and the spots with special colors after the development are black brown spots after the development with 10% sulfuric acid-ethanol solution.
Further, component A1 in step (3) is purified by liquid chromatography TM RP18 chromatographic column (4.6 mm ×250 mm,5 μm) at 35 deg.C and methanol-water 70:30 to obtain the target component compound monomer.
The invention discloses application of a pinane diterpene with a structural formula shown in a formula I in preparation of anti-inflammatory drugs.
Further, anti-inflammatory activity is achieved by reducing NO release in the cell.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a new component obtained by using the compound with a new structure, which defines the extraction and separation and structure identification methods of the new component, and adopts separation methods such as reflux extraction, solvent extraction, MCI, silica gel column, HPLC chromatography and the like, thereby having simple method and being capable of repeatedly and continuously obtaining the component. Meanwhile, the component shows a certain in-vitro anti-inflammatory activity, and provides a material basis for the research and development of new anti-inflammatory drugs.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
Fig. 1: critical HMBC (% and) of compounds 1 H- 1 H COSY (━) panels.
Fig. 2: the effect of the compound on RAW264.7 cell viability and the inhibition of NO release (n=3, MTT assay corresponds to the right ordinate, in% and NO assay corresponds to the left ordinate, in. Mu. Mol/L; diDOX: positive control, C: blank control, LPS: LPS addition).
Fig. 3: HR-ESI-MS of a Compoundm/z。
Fig. 4: of compounds 1 H NMR spectrum.
Fig. 5: of compounds 13 C NMR spectrum.
Fig. 6: HMBC spectra of compounds.
Fig. 7: HSQC spectrum of the compound.
Fig. 8: of compounds 1 H- 1 H COSY profile.
Fig. 9: NOESY spectra of the compounds.
Fig. 10: key NOESY («) profile of compounds.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. The singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
1 instrument, reagent and materials
1.1 Instrument and reagent
Bruker Avance III 600 Nuclear magnetic resonance spectrometer (Bruker, switzerland); METTLER TOLEDO XSE205 and 205; a switzerland Buchi R210 rotary evaporator; DP series oilless vacuum pump: shi take advantage of the world science and technology (Tianjin) limited company; agilent 1200 HPLC chromatograph; HPLC-IT-TOF-MS liquid chromatography-mass spectrometry (Shimadzu corporation, japan); bio Tek EPOCH2NS microplate reader.
Reagents such as diethyl ether, n-butanol, ethyl acetate, methanol, ethanol, petroleum ether, acetone, methylene dichloride and the like are all analytically pure; MCI GEL CHP20P: 37-75 μm, mitsubishi chemical corporation, japan; column layer chromatography silica gel: 200-300 mesh, qingdao Bay Fine chemical Co., ltd; sephadex LH-20 (Sephadex LH-20) Beijing Soy Bao technology Co., ltd; normal phase silica gel thin layer plate: the institute of chemical industry in the tobacco stand market; MTT solution: beijing Ding Guo prosperous biotechnology limited company; (Eagle) DMEM high sugar medium: gibco; fetal bovine serum: shanghai Datthe Biotech Co., ltd; dimethyl sulfoxide (DMSO): LIFE SCIENCE company; LPS lipopolysaccharide: sigma Aldrich(Shanghai) trade Limited; para-aminobenzenesulfonamide: beijing Ding Guo prosperous biotechnology limited company; pancreatic enzyme: beijing Ding Guo prosperous biotechnology limited company; DIDOX: MCE. Cortex Acanthopancis is obtained from Hubei, and is a plant of AraliaceaeAcanthopanax gracilistylusW.w. Smith dried root.
2 Experimental methods
2.1 Preparation of crude extract
Pulverizing the collected cortex Acanthopanacis medicinal material 4 Kg, placing into a 5L round bottom flask, extracting with 70% ethanol for 3 times by adopting a heating reflux extraction mode, each time 1 h, mixing the extractive solutions, and recovering solvent under reduced pressure and rotary steaming at 57 ℃ until the obtained solution is odorless, thus obtaining ethanol crude extract 600 g.
2.2 Graded extraction
Dispersing the crude extract with warm water, extracting with diethyl ether, standing to completely delaminate, separating the extraction solvent, repeating extraction for 3 times, mixing the extracts, and concentrating under reduced pressure to obtain diethyl ether layer extract 130.2 g.
2.3 Separation and purification
Dissolving the ether layer extract, then passing through an MCI resin column, sequentially carrying out gradient elution by 40% -100% methanol to obtain 7 fractions, wherein the fraction 4 is subjected to forward silica gel chromatographic column, gradient elution by petroleum ether-acetone system (100:1-1:1), and detection by a thin-layer silica gel plate, and combining the same components to obtain Fr. A-X total 24 components. The target compound is obtained by separating the component J through liquid chromatography (column temperature: 35 ℃ C., methanol-water 70:30).
2.4 identification of chemical Components
2.4.1 Nuclear magnetic resonance analysis
And taking a proper amount of the separated compounds, respectively dissolving the compounds by using deuterated chloroform, and then placing the compounds into a nuclear magnetic tube, and measuring the compounds by using a Bruker Avance III 600 nuclear magnetic resonance spectrometer.
2.4.2 Mass spectrometry analysis
A small amount of the test compound was taken and dissolved in methanol and analyzed by a mass spectrometer. Electrospray ion source (ESI), sample injection flow rate is set to be 0.3 mu L/h, and detection mode positive and negative ion switching is carried out simultaneously for detection: scanning rangem/z50-1500; ion accumulation time50 ms; the flow rate of the atomized gas (nitrogen) is 1.5L/min; the temperature of the curve desolventizing pipe and the temperature of the heating module are 200 ℃; CID energy 50%; detector voltage, 1.69 kV; the dry gas (nitrogen) pressure was 111 kPa.
2.5 anti-inflammatory Activity Studies
2.5.1 RAW264.7 cell culture
At 37℃with 5% CO 2 In the environment, fetal bovine serum was added to the medium cultured in high-sugar DMEM, and resuscitated RAW264.7 cells were added. Taking cells in logarithmic growth phase, counting and diluting the cell density to 8×10 5 mu.L. Add 96-well plates, 200 μl per well.
2.5.2 MTT method for detecting influence of drug on RAW264.7 cell viability
Regulating cell concentration of logarithmic growth phase cells to 8×10 5 mu.L, 200. Mu.L per well, were inoculated into 96-well plates, and the group treatment was performed as a blank control group, a positive control group (DiDO: 3, 4-dihydroxybenzoic hydroxamic acid), and an experimental group. After the cells added with the drugs are respectively cultured for 24 h, 20 mu L of MTT solution is added into each hole, the culture is continued for 2 h, the supernatant is removed, 200 mu L of DMSO is added and vibrated away from light for 10 min, the Optical Density (OD) value of each hole is detected at 570 nm wavelength by adopting a full-wavelength enzyme-labeled instrument, and the cell survival rate is calculated.
2.5.3 testing of the monomeric Compounds for the release of NO from RAW264.7 cells
Taking RAW264.7 cells in logarithmic growth phase, and adjusting cell concentration to 2×10 5 Per mL, 200. Mu.L per well in 96-well plates at 37℃with 5% CO 2 After 24. 24 h culture in the environment, the supernatant was removed and the cells were divided into a blank (blank medium), a lipopolysaccharide (5. Mu.g/mL lipopolysaccharide), and compounds of different mass concentrations (1.0. Mu.g/mL lipopolysaccharide+100, 50, 25, 12.5, 6.25, 3.125. Mu.g/mL). After each group of cells was added with drug or blank medium, the culture was continued for 24 h. Taking 100 mu L of supernatant, adding 100 mu L of Griess reagent, standing at room temperature for 10 min, setting the wavelength to 570 nm, measuring absorbance on a microplate reader, substituting the absorbance into a standard curve, and calculating the NO release amount.
3 results and analysis
3.1 isolation results
Separating and purifying the diethyl ether layer extract by MCI resin column, silica gel column chromatography, and thin layer plate preparation separation method and thin layer chromatography detection analysis to obtain the target compound 3.3 mg. The structural formula is shown as a formula I,
。
3.2 structural identification of Compounds
White amorphous powder, easily dissolved in methanol, developed with a thin layer of petroleum ether-ethyl acetate (7:3), had dark spots under 254 nm, fluorescence under 365 nm, and developed black brown spots after heating by spraying 10% sulfuric acid-ethanol. HR-ESI-MSm/ z) 287.1641 [M-H] - (FIG. 3), theoretical value 287.1653, deviation 4.2 ppm, and its molecular formula C 18 H 24 O 3 The unsaturation was 7. 1 H NMR (FIG. 4, table 1) shows that there are 2 angular methyl groups [δ H 1.12(s), 1.27(s)]And 1 double bond-linked methyl groupδ H 2.14(s)]Is present. By HSQC (FIG. 7) and 13 c NMR (FIG. 5)δ C 122.7 (C-8), 147.2 (C-9), 116.6 (C-11), 127.4 (C-12), 120.3 (C-13), 151.7 (C-14) 6C signals can determine the presence of a benzene ring,δ H 6.73(J=8.4 Hz) andδ H 6.84(J=8.4 Hz) are two adjacent hydrogen protons on the benzene ring. By HMBC (FIG. 6)δ H 6.84(J=8.4 Hz) is correlated with the C-9, C-11 and C-14 signals,δ H 6.73(J=8.4 Hz) with C-8, C-9 and C-13 signals, it was determined that the phencyclized hydrogens are at the 11 and 12 positions; the hydroxyl group at the 14 position can be deduced through the related signals of C-14, H-7 and H-15 in HMBC; by H-16 in HMBCδ C 180.1 the correlation may speculate that the carboxilic group is at position 17. According to HMBCδ C 147.2 to H-7, H-12, H-18,δ C 122.7 to H-6, H-7, H-11, H-14,δ C the correlation of 38.1 with H-1, H-2, H-5, H-18 can further confirm its structure. According to 1 H- 1 Correlation of H-1 with H-2, H-2 with H-3, H-5 with H-6, H-6 with H-7, H-11 with H COSY (FIG. 8)H-12, in combination with information in HMBC, can determine its planar structure (see FIG. 1). By the NOESY (FIG. 9) spectra of H-5 and H-1β、H-7βH-16, and deducing its relative configuration (see FIG. 10). Thus, the structure of the compound was identified as 14-hydroxy-16-de-ethenyl-enantiomer-pimo-8, 11, 13-triene-17-carboxylic acid.
The compounds of Table 1 1 H and 13 c NMR data
| Position of | δ H | δ C |
| 1α | 2.23(m) | 39.7 |
| 1β | 1.31(m) | |
| 2α | 1.98(m) | 19.9 |
| 2β | 1.56(m) | |
| 3α | 2.21(m) | 37.5 |
| 3β | 1.07(dt,J= 13.2, 4.2 Hz) | |
| 4 | 43.5 | |
| 5 | 1.47(d,J= 12.6 Hz) | 52.5 |
| 6α | 2.19(m) | 20.4 |
| 6β | 1.96(m) | |
| 7α | 2.90(dd,J= 16.8, 5.4 Hz) | 25.8 |
| 7β | 2.45(m) | |
| 8 | 122.7 | |
| 9 | 147.2 | |
| 10 | 38.1 | |
| 11 | 6.73(d,J= 8.4 Hz) | 116.6 |
| 12 | 6.84(d,J= 8.4 Hz) | 127.4 |
| 13 | 120.3 | |
| 14 | 151.7 | |
| 15 | 2.14(s) | 14.7 |
| 16 | 1.27(s) | 27.9 |
| 17 | 180.1 | |
| 18 | 1.12(s) | 22.3 |
Note that: 1 h NMR 13 C NMR was 600 and 150 MHz respectively; the solvent is CD 3 OD。
Note that: 1 h NMR 13 C NMR was 600 and 150 MHz respectively; the solvent is CD 3 OD。
3.3 screening results for anti-inflammatory Activity
3.3.1 Effect of monomer Compounds on cell RAW264.7 cell viability
Cells were divided into a blank control group, a positive control group (DiDO: 3, 4-dihydroxybenzohydroxamic acid) and an experimental group. Compared with a blank control group, the compound has the drug concentration within the range of 3.125-100 mug/ml, is nontoxic to cells, and has the cell activity of more than 80%. The results are shown in FIG. 2.
3.3.2 determination of NO content in LPS stimulated RAW264.7 cells by monomeric Compounds
Cells were divided into a blank (blank medium, without lipopolysaccharide LPS), a lipopolysaccharide group (5. Mu.g/mL lipopolysaccharide LPS), a positive control (1.0. Mu.g/mL lipopolysaccharide+100, 50. Mu.g/mL 3, 4-dihydroxybenzoic hydroxamate DiDO), compounds of different mass concentrations (1.0. Mu.g/mL lipopolysaccharide+100, 50, 25, 12.5, 6.25, 3.125. Mu.g/mL compounds). As a result, as shown in FIG. 2, the compound had a certain anti-inflammatory activity at 50, 100. Mu.g/ml, and the amount of NO released decreased with increasing concentration, as compared with LPS group. At a concentration of 100. Mu.g/ml, the anti-inflammatory activity was comparable to that of the positive drug DiDO 50. Mu.g/ml.
The anti-inflammatory activity shows that the compound has weaker anti-inflammatory activity, and can obviously inhibit NO release in RAW264.7 cells induced by LPS when reaching a certain concentration (more than 50 mu g/ml), thereby inhibiting NO-mediated inflammatory reaction. Meanwhile, experiments show that the component has no toxicity to cells, so that the component can be developed into an anti-inflammatory drug.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A kind of sea pine alkane diterpene is characterized in that the structural formula is shown in the formula I,
。
2. the process for the preparation of a norpimaran diterpene of formula I of claim 1, characterized by the steps of:
(1) Preparing a crude extract: pulverizing cortex Acanthopanacis, extracting with 70% ethanol under reflux, collecting polar part, and steaming at 60deg.C under reduced pressure to obtain crude extract;
(2) And (3) carrying out fractional extraction: dispersing the crude extract with warm water, extracting with diethyl ether, standing to completely layer, separating the extraction solvent, repeatedly extracting, mixing the extractive solutions, and concentrating under reduced pressure to obtain diethyl ether layer extract;
(3) And (3) separating and purifying: separating the ether layer extract by using an MCI resin column, performing gradient elution by using a methanol aqueous solution, wherein the fraction A passes through a forward silica gel chromatographic column, performing gradient elution by using a petroleum ether-acetone system, detecting by using a thin-layer silica gel plate, combining the same components, targeting to find spots with dark spots and fluorescence under an ultraviolet lamp in the silica gel thin-layer chromatography, and separating the component A1 with the target spots by using a liquid chromatography to obtain a target compound;
(4) Identification of chemical components: the chemical structure of the target compound is determined using high resolution mass spectrometry, 1D and 2D nmr spectroscopy.
3. The process according to claim 2, wherein the reduced pressure rotary evaporation temperature in step (1) is 57 ℃.
4. The method according to claim 2, wherein the MCI resin column in step (3) is eluted with a 40% -100% aqueous methanol gradient to separate the target component from other interfering components.
5. The preparation method according to claim 2, wherein the fraction A in the step (3) is subjected to forward silica gel chromatographic column, and is subjected to gradient elution by a petroleum ether-acetone system, and the volume ratio of petroleum ether to acetone is 100:1-1:1, so that the target component is further separated from other interference components.
6. The method according to claim 2, wherein the dark spots observed in the step (3) have a wavelength of 254.+ -.2 nm and a fluorescence observed wavelength of 365.+ -.2 n, and the spots having a specific color after the development are spots which develop a black brown color in sunlight after the development with a 10% sulfuric acid-ethanol solution.
7. The process according to claim 2, wherein component A1 in step (3) is purified by liquid chromatography TM RP18 chromatographic column with temperature of 35 deg.C and methanol-water separation of 70:30 to obtain the target component compound monomer.
8. Use of a norpimaran-type diterpene of formula I as claimed in claim 1 for the manufacture of an anti-inflammatory agent whose anti-inflammatory activity is achieved by reducing NO release in cells.
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| EP3245184A1 (en) * | 2015-01-12 | 2017-11-22 | Elinder, Fredrik | Dehydroabietic acid (dhaa) derivatives for use as ion channel openers |
| CN109908133A (en) * | 2019-04-04 | 2019-06-21 | 山东大学 | A kind of pimarane type diterpene is preparing the application in anti-oxidation stress medicine |
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