CN108640968A - A kind of meroterpenoids compound and its purposes in preparing anti-inflammatory drug - Google Patents
A kind of meroterpenoids compound and its purposes in preparing anti-inflammatory drug Download PDFInfo
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- CN108640968A CN108640968A CN201810595613.2A CN201810595613A CN108640968A CN 108640968 A CN108640968 A CN 108640968A CN 201810595613 A CN201810595613 A CN 201810595613A CN 108640968 A CN108640968 A CN 108640968A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J73/00—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms
- C07J73/001—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom
- C07J73/003—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom by oxygen as hetero atom
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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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
Abstract
The present invention relates to a kind of meroterpenoids compound and its purposes in preparing anti-inflammatory drug.The structure of the meroterpenoids compound is shown in formula I, and preparation method is that marine source Aspergillus terreus 44 fermentates of ML are extracted, isolates and purifies to obtain.The present invention has the function of inhibiting 264.7 cell NO releases of LPS inductions RAW, the effect is realized not by inhibition cell Proliferation or toxicity, show with anti-inflammatory activity, therefore can be used as anti-inflammatory drug for treating diseases associated with inflammation, lead compound is provided for exploitation novel anti-inflammatory drug.
Description
Technical field
The present invention relates to a kind of meroterpenoids compound and its purposes in preparing anti-inflammatory drug.
Background technology
The natural products that mixed source terpene is used to describe to be formed by isopentene group approach heterozygosis other biological route of synthesis, has
Structure diversity complicated and changeable, and then show diversified bioactivity.Partly such compound has been used as medicine
Object is for clinic, such as antitumor drug vinblastine, immunosuppressor mycophenolate, acetylcholinesterase inhibitor soil shake element B
Deng.Terretonins is one kind with 3,5-demethylorsellinic acid（DMOA）And arnesyl
pyrophosphate（FPP）To synthesize the originated from fungus meroterpenoids compound of precursor, mainly divide from Aspergillus tunning
From obtaining（Guo et al., Molecular genetic characterization of a cluster inA. terreus for biosynthesis of the meroterpenoid terretonin. Org Lett. 2012, 14:
5684–5687）.Up to now, Terretonin（Springer, Terretonin, a toxic compound fromAspergillus terreus. J Org Chem, 1979, 44: 4852–4854）And its analogue
terretonins A–D（Li et al., Sesterterpenoids, terretonins A-D, and an
alkaloid, asterrelenin, from Aspergillus terreus. J Nat Prod, 2005, 68:1243–
1246）、H–K（Matsuda et al., Uncovering the unusual D-ring construction in
terretonin biosynthesis by collaboration of a multifunctional cytochrome P450
and a unique Isomerase. J Am Chem Soc, 2015, 137:3393–3401）And M（Shaaban et al.
Terretonin M: A new meroterpenoid from the thermophilic Aspergillus terreus
TM8 and revision of the absolute configuration of penisimplicins. Nat Prod
Res, 2017, doi: 10.1080/14786419.2017.1419230）It is isolated from Aspergillus terreusAspergillus terreusIn fermentate, there are the unique parallel tetracyclic structure parent nucleus shown in Formulas I in molecule, and natural products is exhausted
Consistent to configuration, wherein 14-H is orientated for β.Two analogues found in the recent period, " terretonin H " and
" terretonin I " is isolated from marine source aspergillus ustus Aspergillus ustus（Oleinikova et al., Two
new sesterterpenoids, terretonins H and I, from the marine-derived fungusAspergillus ustus. Phytochem Lett, 2016, 17:135–139）, its structure is special compared with above compound
Sign is that C-14 absolute configurations change, i.e. 14-H is orientated for α.Terretonins E and F presses down as mitochondrial respiratory chain
Preparation is isolated from marine-derived fungal Aspergillus insuetus fermentates（Lopez-Gresa et al.,
Terretonins E and F, inhibitors of the mitochondrial respiratory chain from
the marine-derived fungus Aspergillus insuetus. J Nat Prod, 2009, 72:1348–
1351）, belong to a kind of special terretonins hypotypes with terretonin L.
Chinese patent CN201510244085.2 reports a kind of α-pyranone and mixes source terpene and its preparation method and application, answers
It is used to prepare anti-influenza virus medicament.CN107362077A only once property refer to Sargassum plant rich in glyceroglycolipid,
The ingredients such as phytosterol, meroterpenoids, brown algae polyphenols, nitrogenous compound have antitumor, anti-oxidant, reducing blood lipid, antiviral etc.
Multiple biological activities.Therefore, so far about terretonins activity research report it is considerably less, have no report terretonin and
Its analog has anti-inflammatory activity.
Invention content
The object of the present invention is to provide a kind of new meroterpenoids compounds and its purposes in preparing anti-inflammatory drug.It should
Meroterpenoids compound and its analogue have anti-inflammatory activity, provide lead compound for exploitation novel anti-inflammatory drug, use
As anti-inflammatory drug for treating diseases associated with inflammation.
Meroterpenoids provided by the invention（terretonins）Compound is as shown in following Formulas I structures：
I
Compound of formula I of the present invention, is different from known compound terretonin D, and 14-H is orientated for α.
The analogue terretonin for further relating to compound of formula I and compound of formula I of the present invention, i.e. compound II,
Terretonin A, i.e. compound III, terretonin D, i.e. compound IV are individually or with composition forms（As pharmaceutically may be used
The adjuvant etc. of receiving）Purposes in preparing anti-inflammatory drug.
II III IV
The present invention provides Aspergillus terreus（Aspergillus terreus）ML-44, deposit number are CGMCC No.
15664, the deposit date is on 04 17th, 2018, depositary institution was China General Microbiological culture presevation administrative center
（CGMCC）, address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101.
The bacterial strain is detached from the alimentary canal sample of Pacific oyster, and categorized research is accredited as Aspergillus terreus（Aspergillus terreus）.
Compound of formula I provided by the invention and its preparation method of analogue include the following steps：
Above-mentioned Aspergillus terreus is subjected to fermented and cultured, the fermentate containing compound of formula I and its analogue is obtained, will ferment
Object is isolated and purified, and the compound is obtained.Other production bacterium for capableing of compound of formula I of aspergillus can also be used
Carry out fermented and cultured.
It is described to isolate and purify the conventional method purified including the use of Separation of Natural Products well known to those skilled in the art, such as
Extraction, column chromatography, efficient liquid phase preparation etc..
The specific preparation method of compound of formula I provided by the invention includes the following steps：
1) above-mentioned Aspergillus terreus is subjected to rice solid fermentation culture, obtains fermentate；
2) by gained fermentate 50%-90%（v/v）Aqueous acetone solution ultrasound extraction, filtering, filtrate is through being concentrated under reduced pressure into not
Containing acetone, remaining suspension is extracted with ethyl acetate, and obtains the acetic acid ethyl ester extract of fermentate；
3) step 2) is obtained acetic acid ethyl ester extract to be concentrated to dryness, obtains ethyl acetate extract；
4) the total medicinal extract of ethyl acetate is used into silica gel column chromatography, petroleum ether dichloromethane methanol gradient elution, Sephadex successively
LH-20 column chromatographies, methanol elution and ODS column chromatographies, the methanol elution gradient of water → 100%, separation are obtained containing the compound
Column chromatography component；
5) by the column chromatography group lease making HPLC separation containing the compound.
Aqueous acetone solution in step 2) is 70%-90%（v/v）, preferably 75%-85%（v/v）, more preferably 80%（v/
v）.
The present invention uses Griess methods, thin to LPS inductions RAW 264.7 to compound of formula I and its above structure analog
The inhibiting effect of born of the same parents' NO releases is tested.It is verified by experiments, compound of formula I and above structure analog can significantly inhibit
LPS induces the release of 264.7 cell NO of RAW, thus has anti-inflammatory effect.
The extract of the fermentate for additionally providing Aspergillus terreus ML-44 of the present invention, the extract contain the Formulas I of the present invention
Compound and its analogue.Specifically, the extract is the acetic acid ethyl ester extract of Aspergillus terreus ML-44 fermentates, second
The total medicinal extract of acetoacetic ester or chromatographic fraction.The extract is referred to the phase of the preparation method of the compound of the present invention above
Step is answered to be made.Specifically, the extract can be prepared by extraction, column chromatography, efficient liquid phase.
The present invention also provides purposes of the extract of Aspergillus terreus ML-44 fermentates in preparing anti-inflammatory drug.
In short, the present invention provides have structure and its above structure analog shown in Formulas I containing described（II-
IV）Or the drug of its pharmaceutically acceptable salt, the drug include pharmaceutically acceptable adjuvant；The drug is injection
Agent, tablet, pill, capsule, suspending agent or emulsion etc..
The meroterpenoids compound or its pharmaceutically acceptable salt individually or with composition forms prepare it is anti-inflammatory
Drug application.
The present invention provides a kind of anti-inflammatory drug, including a effective amount of meroterpenoids compound or its pharmaceutically
Acceptable salt and pharmaceutically acceptable carrier.
The present invention also provide above-mentioned Aspergillus terreus ML-44 in the compound of formula I for preparing the present invention or fermentate carries
Take the purposes in object.
The compound of formula I and its above structure analog of the present invention can be with various pharmaceutically acceptable carriers, excipient
Or anti-inflammatory drug is made in supplementary product compatibility, is used for the treatment of diseases associated with inflammation.
The compounds of this invention can be administered individually or in the form of pharmaceutical composition.Administration route can be take orally, non-bowel
Or local administration.Pharmaceutical composition can be made into various suitable dosage forms according to administration route.
The pharmaceutical composition of the compounds of this invention can be applied with following any way：Oral, spraying sucking, rectum is used
Medicine, nasal cavity applied medicine, cheek medication, local application, non-bowel medication, such as subcutaneous, vein is intramuscular, intrathecal in peritonaeum, intra-ventricle,
In breastbone and intracranial injection or input, or by a kind of explant reservoir medication.It wherein preferably takes orally, peritonaeum is interior or intravenous administration
Mode.
When oral medication, the compounds of this invention, which can be made into, arbitrarily takes orally acceptable dosage form, including but not limited to
Tablet, capsule, aqueous solution or water slurry.Wherein, the carrier that tablet uses generally comprises lactose and cornstarch, in addition also may be used
Lubricant such as magnesium stearate is added.The diluent that capsule preparations use generally comprises lactose and dried corn starch.Water slurry
Preparation is typically then to be used in mixed way active constituent and suitable emulsifier and suspending agent.Optionally, the above oral dosage form
In some sweeteners, aromatic or colorant can also be added.
When topical application, the compounds of this invention can be made into ointment, lotion or cream formulation form appropriate, wherein
Active constituent is suspended or dissolved in one or more carriers.Carrier workable for ointment formulation includes but not limited to：Mineral
Oil, Albolene, albolene, propylene glycol, polyethylene glycol oxide, polypropylene oxide, emulsifying wax and water；Lotion or creme can make
Carrier includes but not limited to：Mineral oil, sorbitan monostearate, polysorbate60, cetyl ester wax, hexadecene
Fragrant and mellow, 2- octyldodecanols, benzyl alcohol and water.
The compounds of this invention can the medication in the form of aseptic injection preparation, including aseptic injection water or oil suspension or sterile
Inject solution.Wherein, workable carrier and solvent include water, Ringer's solution and isotonic sodium chlorrde solution.In addition, sterilizing
Fixed oil also is used as solvent or suspension media, such as monoglyceride or two glyceride.
Furthermore, it should be pointed out that the compounds of this invention dosage and application method depend on factors, including patient
Age, weight, gender, natural health situation, nutrition condition, the activity intensity of compound, Time of Administration, metabolic rate, illness
Severity and diagnosis and treatment doctor subjective judgement.Preferred dosage is between 0.01-100 mg/kg body weight/days.
The present invention provides compound of formula I and its above structure analogs（II- IV）Cell activity test, the present invention
Have the function of inhibiting 264.7 cell NO releases of LPS inductions RAW, the effect real not by inhibition cell Proliferation or toxicity
Existing, show with anti-inflammatory activity, therefore can be used as anti-inflammatory drug for treating diseases associated with inflammation, to develop novel anti-inflammatory medicine
Object provides lead compound.
Description of the drawings
Fig. 1 is the X-ray single crystal diffraction structure of compound I.
Fig. 2 is the HR-ESI-MS spectrograms of compound I.
Fig. 3 is compound I's1H NMR spectras.
Fig. 4 is compound I's13C NMR spectras.
Fig. 5 is the DEPT spectrograms of compound I.
Fig. 6 is compound I's1H-1H COSY spectrograms.
Fig. 7 is the HMQC spectrograms of compound I.
Fig. 8 is the HMBC spectrograms of compound I.
Fig. 9 is the NOESY spectrograms of compound I.
Specific implementation mode
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products obtained can be bought by market.
In the examples below, referred to as compound I is compound shown in Formulas I, be present invention firstly discovers that new chemical combination
Object；That referred to as compound II is known compound terretonin；That referred to as compound III is known compound terretonin
A；That referred to as compound IV is known compound terretonin D.
Arabic numerals indicate corresponding mark.
I
In the structural research of embodiment below, specific rotatory power is measured with Japan's JASCO P-1020 type polarimeters.ESI-MS and
HR-ESI-MS uses Thermo Scientific companies of U.S. LCQ Fleet LC-MS instrument and Agilent companies LCT respectively
6200 series TOF/6500 type mass spectrographs measure, NMR spectra 500 type superconduction cores of Bruke companies of Switzerland Avance III
Magnetic resonance device（500 MHz1H-NMR, 125 MHz13C-NMR）It measures.
Embodiment 1：The preparation of microbial fermentation culture and compound
1. the extraction process of fermented and cultured and fermentate
1) bacterial strain is produced
Producing strains in the present embodiment for fermenting and producing compound I-IV are to be isolated from the gastral Aspergillus terreus of Pacific oyster
Bacterium（Aspergillus terreus）ML-44 plants, it is preserved in China General Microbiological culture presevation administrative center（CGMCC）, protect
It is CGMCC No. 15664 to hide number.
2) fermented and cultured
By the conventional method of microculture, Aspergillus is taken out from 4 DEG C of refrigerators（Aspergillus terreus）ML-44 test tubes
Inclined-plane aseptically scrapes appropriate spore with oese, and streak inoculation is in the 5 PDA solid mediums newly prepared（Group
At：Glucose 2%, agar 2%, NaCl 1.5% are prepared with the water cooking liquid of 20% potato）On tablet, training is activated in 28 DEG C of incubators
It supports 5 days, takes spore with the sterile washings of 10ml respectively, and all spores merging is scattered in 100 ml sterile waters, obtain spore
Suspension.The inoculum concentration that the spore suspension is pressed to every bottle of 4 ml, is inoculated in respectively equipped with rice solid medium（Every bottle of 80 g are commercially available
Rice adds 150 ml natural sea-waters, is made in 121 DEG C of 30 min of sterilizing after placement is overnight）24 1000 ml conical flasks in,
It is cultivated 35 days in being stored at room temperature.
2. the preparation of extraction process and ethyl acetate extract
The aqueous acetone solution for being 80% by fermentate volume fraction（Every bottle of 500 ml）It impregnates and is suspended, be ultrasonically treated 1h, room temperature
Extraction is for 24 hours afterwards with 4 layers of filtered through gauze, repetitive operation 3 times.Merging filtrate is concentrated under reduced pressure into without after acetone, remaining aqueous layer about 3L
It is extracted 3 times with isometric ethyl acetate, obtains fermentate acetic acid ethyl ester extract, be concentrated to dryness, obtain ethyl acetate extract
66.2 g。
3. the preparation of the column chromatography for separation of ethyl acetate extract and the column chromatography component containing target compound I-IV
By 66.2 g methylene chloride-methanols of above-mentioned ethyl acetate extract（v/v 1:1）200ml dissolves, and filters out insoluble matter, is added
90 g chromatographic silica gels（200-300 mesh）Sample is mixed in absorption, is added to prepackage silica gel glass decompression column after evaporated under reduced pressure（120 g silica gel,
4.8 × 18.0 cm of column bed）On, gradient elution chromatography is carried out with petroleum ether-methylene chloride-methanol solvent system, is obtained several
Flow point.Merge corresponding flow point according to silica gel thin-layer analysis result, 8 components are obtained by elution order of polarity：Fr-1（12.4 g,
Petroleum ether）、Fr-2（6.3 g, petroleum ether-dichloromethane 1:1 elution）、Fr-3（4.5 g, petroleum ether-dichloromethane 1:2 wash
It is de-）、Fr-4（11.6 g, dichloromethane eluent）、Fr-5（15.8 g, methylene chloride-methanol 99:1 elution）、Fr-6（6.2 g,
Methylene chloride-methanol 95:5 elutions）、Fr-7（1.4 g, dichloromethane 9:1 elution）、Fr-8（1.9 g, methylene chloride-methanol 7:
3 elutions）.
By component Fr-3（4.5 g）It is dissolved with 50 ml methanol, is loaded to the Sephadex LH-20 columns of methanol prepackage（Column
4.8 × 42 cm of bed）, methanol elution chromatography, by elution sequencing fraction is merged into 4 sub- components：Fr-3-1-Fr-3-
5.Fr-3-3（1.1 g）After being dissolved with methanol and used 0.45 μm of membrane filtration, using QuikSep mesolow chromatographic systems（Intelligent moral
Easily, Beijing, China）With 80% methanol（10 ml/min of flow velocity, room temperature）In the glass column for being preinstalled with ODS（Column bed 1.8cm ×
30cm）On detached.5 components are obtained according to ultraviolet testing result：Fr-3-3-1-Fr-3-3-5.Wherein, in Fr-3-3-1
Contain target compound I-IV.
4. prepared by the HPLC of compound I-IV
By the component Fr-3-3-1 containing compound I-IV（570 mg）It is dissolved with 10 ml methanol, after 0.45 μm of membrane filtration,
With 2545 type HPLC systems of Waters, column is prepared using Gemini C18（21.2 mm × 250 mm）Carry out HPLC separation
（26 DEG C of column temperature, using 75% methanol as mobile phase, 10 ml/min of flow velocity, 0.5 ml of each sample introduction, Detection wavelength are 210 and 254
Nm), compound I is made（28 mg,t R= 17.3 min）, compound II（118 mg,t R= 12.9 min）, compound III
（85 mg,t R= 26.8 min）With compound IV（130 mg,t R= 24.9 min）.
The physicochemical constant and spectral data of compound I-IV
Compound I is colourless lump shaped crystalline（MeOH）, m.p. 172-174oC, [α] -86.8o (c1.0, MeOH).UV
(MeOH)：End absorbs.Cation ESI-MS：m/z 475 [M + H]+, 497 [M+Na]+, 971 [2M+Na]+；It is negative
Ion ESI-MS：m/z 491 [M + H2O - H]-, 509 [M+Cl]-.HR-ESI-MS：m/zMeasured value 475.2332
[M + H]+, calculated value C26H35O8 [M + H]+475.2324；Measured value 497.2151 [M+Na]+, calculated value
C26H34O8Na [M + Na]+497.2146；Measured value 509.1942 [M+Cl]-, calculated value C26H34O8Cl [M + Cl]-
509.1950.NMR data is as shown in Table 1 and Table 2.
The nuclear magnetic data of compound I is belonged to through the analysis of the two-dimensional maps such as COSY, HSQC and HMBC, releases its planar junction
Structure and compound IV（terretonin D）Unanimously；Its absolute configuration is through NOESY spectrum analysis and X-ray single crystal diffraction analysis mirror
It is set to epimers of the terretonin D at C-14.
1 compound I-IV's of table1H NMR datas（500 MHz, CDCl3）
Compound II is white powder（MeOH）, [α] -96.8 (c1.0, MeOH).Cation ESI-MS：m/z 489
[M + H]+, 511 [M+Na]+, 999 [2M+Na]+；Anion ESI-MS：m/z 487 [M - H]-。1H and13C NMR
Data are as shown in Table 1 and Table 2.
Compound III is white powder（MeOH）, [α] -112.5 (c1.0, MeOH).Cation ESI-MS：m/z
473 [M + H]+, 495 [M+Na]+, 967 [2M+Na]+；Anion ESI-MS：m/z 471 [M - H]-.NMR numbers
According to as shown in Table 1 and Table 2.
Compound IV is white powder（MeOH）, [α] -63.4 (c1.0, MeOH).Positive ESI-MS:m/ z 475 [M + H]+, 497 [M + Na]+, 971 [2M + Na]+; negative ESI-MS: m/z 473 [M -
H]-, 509 [M + Cl]-NMR data is as shown in Table 1 and Table 2.
2 compound I-IV's of table13C NMR datas（125 MHz, CDCl3）
Embodiment 2：The anti-inflammatory activity of compound I-IV is tested
1. experiment material
1) preparation of sample solution
Test sample is the sterling compound I-IV of separation and purification in above-described embodiment 1.Precision weighs appropriate amount of sample, is matched with methanol
At the solution of required concentration, for test activity.
2) squamous subculture of cell line and cell
Active testing uses 264.7 cell lines of mouse macrophage RAW.264.7 cells of RAW use containing 10% fetal calf serum and
The DMEM culture medium routine passages of penicillin and each 100 μ g/ml of streptomysin, and trained in 37 DEG C of cells for being passed through 5% carbon dioxide
It supports to cultivate in case and safeguard.
2. activity test method
1) inhibiting effect of the compound to 264.7 cell NO releases of LPS inductions RAW
Using Griess methods, compound I-IV surveys the inhibiting effect of 264.7 cell NO releases of LPS inductions RAW
Examination.264.7 cells of RAW of logarithmic growth phase, it is 2 × 10 to prepare cell density with fresh DMEM medium5A/ml's is thin
Born of the same parents' suspension is inoculated in 96 orifice plates, and sample sets add each 2 μ l of sample liquid, blank per hole after every hole 200 μ l, 37 DEG C of 12 h of culture
Control group then adds each 2 μ l of methanol per hole, handles 1 h in 37 DEG C, LPS is then added per hole（1 μg/mL）, continue to cultivate 24 h.
After culture, take 100 μ l of supernatant per hole, respectively with isometric Griess reagents（1% sulfanilamide (SN), 0.1% hydrochloric acid naphthalene second two
Amine and 2.5% phosphoric acid）Mixing, measures absorbance value of each hole at 540 nm after reaction（OD values）.By formula IR%=(ODLPS-
ODSample)/(ODLPS-ODBlank) × 100% calculates NO and generates inhibiting rate（IR%）.
2) influence of the compound to 264.7 cell viabilities of RAW
Influence using mtt assay detection compound to 264.7 cell viabilities of RAW.100 μ l supernatants are taken out for measuring NO water
After flat, the MTT solution of the 5mg/ml of precooling is added per hole（It is prepared with PBS solution）Each 20 μ l, after 37 DEG C are incubated 4 h, in 4
DEG C, 2000 rpm centrifuge 10 min, suck supernatant, add 150 μ l DMSO per hole, being placed in microplate reader fully oscillation makes
MTT purple products are completely dissolved, and measure the OD values at per 570 nm of hole.Sample and blank control group respectively set three in experiment
Parallel hole takes OD average values, by IR%=(ODBlank-ODSample)/ODBlank× 100% formula calculates sample to 264.7 cells of RAW
Inhibiting rate（IR%）.
3. experimental result
1) mtt assay test result
In mtt assay test, in conjunction with micro- sem observation inspection, compound I-IV is under 100 μ g/ml activities to RAW
264.7 cells do not show cytotoxicity and inhibiting effect（Table 3）.
2) Griess methods test result
Experimental result shows compound I-IV under 50 μ g/ml activities to 264.7 cell NO release tools of LPS inductions RAW
There is significant inhibiting effect（Table 4）.
4. conclusion
Compound I-IV all has the effect for inhibiting 264.7 cell NO releases of LPS inductions RAW, and the effect is not by inhibition
What cell Proliferation or toxicity were realized, show that compound I-IV has anti-inflammatory activity, therefore compound I-IV can be used as anti-inflammatory agent
Object is for treating diseases associated with inflammation.
Although the specific implementation mode of the present invention has obtained detailed description, it will be understood to those of skill in the art that root
According to all introductions having disclosed, those details can be carry out various modifications and be replaced, these change the guarantor in the present invention
Within the scope of shield.The full scope of the present invention is given by the appended claims and any equivalents thereof.
Claims (10)
1. a kind of being used for anti-inflammatory meroterpenoids compound, it is characterised in that have structure as follows：
I
And
II III IV。
2. the preparation method of meroterpenoids compound described in claim 1, it is characterised in that it is from Aspergillus terreus
（Aspergillus terreus）Preparative separation obtains in the fermentation culture medium of ML-44, is as follows：
1）By Aspergillus terreus（Aspergillus terreus）ML-44 carries out fermented and cultured, obtains fermentate；
2）Fermentate is extracted with ethyl acetate, ethyl acetate extract is obtained；
3）By step 2）In the obtained total medicinal extract of ethyl acetate use successively silica gel column chromatography, Sephadex LH-20 column chromatographies and
ODS column chromatography for separation obtains the column chromatography component containing the compound；
4）By the column chromatography group lease making HPLC purifying containing the compound.
3. preparation method according to claim 2, it is characterised in that prepare Aspergillus terreus（Aspergillus terreus）
The step of fermentation culture medium of ML-44 is：By the conventional method of microculture, from Aspergillus（Aspergillus terreus）The test tube slants ML-44 aseptically scrape appropriate spore with oese, and streak inoculation is in 5 newly prepared
In PDA solid medium tablets, wherein the quality group of solid medium becomes：Glucose 2%, agar 2%, NaCl 1.5% are used
The water cooking liquid of 20% potato is prepared, activation culture 5 days in 28 DEG C of incubators, takes spore with the sterile washings of 10ml respectively, and will be whole
Spore merging is scattered in 100 ml sterile waters, obtains spore suspension；The spore suspension is pressed to the inoculum concentration of every bottle of 4 ml, respectively
It is inoculated in 24 1000 ml conical flasks equipped with rice solid medium, is cultivated 35 days in being stored at room temperature；Rice solid is trained
Supporting the quality group of base becomes：Every bottle of commercially available rice of 80 g adds 150 ml natural sea-waters, and sterilize after placement is overnight in 121 DEG C 30 min
It is made.
4. the fermentate that the preparation method described in claim 3 obtains.
5. Aspergillus terreus（Aspergillus terreus）ML-4 is in preparing meroterpenoids compound described in claim 1
Using.
6. meroterpenoids compound described in claim 1 or its pharmaceutically acceptable salt are being made individually or with composition forms
The application of standby anti-inflammatory drug.
7. a kind of anti-inflammatory drug, it is characterised in that including a effective amount of meroterpenoids compound described in claim 1 or its
Pharmaceutically acceptable salt and pharmaceutically acceptable carrier.
8. according to claim 6 in the application for preparing anti-inflammatory drug, it is characterised in that the drug includes pharmacy
Upper acceptable adjuvant.
9. according to claim 6 in the application for preparing anti-inflammatory drug, it is characterised in that the drug be injection,
Tablet, pill, capsule, suspending agent or emulsion.
10. Aspergillus terreus ML-44, deposit number is CGMCC No.15664.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110028500A (en) * | 2019-04-09 | 2019-07-19 | 嘉兴市爵拓科技有限公司 | Apparent modification Aspergillus terreus secondary metabolites and its purposes in the preparation of antitumor drugs |
| CN112159376A (en) * | 2020-09-24 | 2021-01-01 | 中国人民解放军海军特色医学中心 | Sesterterpene compound and application thereof in preparing anti-inflammatory drugs |
| CN113621526A (en) * | 2021-09-07 | 2021-11-09 | 鲁东大学 | Marine fungus Aspergillus versicolor M-7-SW9, mixed source terpenoid and extraction method and application thereof |
| CN115894408A (en) * | 2021-08-17 | 2023-04-04 | 周口师范学院 | Triisopropenyl substituted aspulvinone compound and preparation method and application thereof |
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2018
- 2018-06-11 CN CN201810595613.2A patent/CN108640968B/en active Active
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110028500A (en) * | 2019-04-09 | 2019-07-19 | 嘉兴市爵拓科技有限公司 | Apparent modification Aspergillus terreus secondary metabolites and its purposes in the preparation of antitumor drugs |
| CN112159376A (en) * | 2020-09-24 | 2021-01-01 | 中国人民解放军海军特色医学中心 | Sesterterpene compound and application thereof in preparing anti-inflammatory drugs |
| CN112159376B (en) * | 2020-09-24 | 2022-04-05 | 中国人民解放军海军特色医学中心 | Sesterterpene compound and application thereof in preparing anti-inflammatory drugs |
| CN115894408A (en) * | 2021-08-17 | 2023-04-04 | 周口师范学院 | Triisopropenyl substituted aspulvinone compound and preparation method and application thereof |
| CN113621526A (en) * | 2021-09-07 | 2021-11-09 | 鲁东大学 | Marine fungus Aspergillus versicolor M-7-SW9, mixed source terpenoid and extraction method and application thereof |
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