CN108277164B - A kind of indene derivatives derived from endophytic fungi in Excoecaria agallocha and its application in preparing anti-inflammatory drugs - Google Patents
A kind of indene derivatives derived from endophytic fungi in Excoecaria agallocha and its application in preparing anti-inflammatory drugs Download PDFInfo
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- 241000233866 Fungi Species 0.000 title claims abstract description 20
- 125000003454 indenyl group Chemical class C1(C=CC2=CC=CC=C12)* 0.000 title claims abstract description 19
- 244000292411 Excoecaria agallocha Species 0.000 title claims abstract description 14
- 229940124599 anti-inflammatory drug Drugs 0.000 title claims abstract description 14
- 230000006698 induction Effects 0.000 claims abstract description 5
- 239000003814 drug Substances 0.000 claims description 7
- 229940079593 drug Drugs 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 abstract description 17
- 239000003973 paint Substances 0.000 abstract description 11
- 239000002260 anti-inflammatory agent Substances 0.000 abstract description 9
- 230000003110 anti-inflammatory effect Effects 0.000 abstract description 7
- 230000002401 inhibitory effect Effects 0.000 abstract description 7
- 241000196324 Embryophyta Species 0.000 abstract description 4
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- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 26
- 210000004027 cell Anatomy 0.000 description 14
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- 239000002158 endotoxin Substances 0.000 description 10
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- 241001373666 Diaporthe sp. Species 0.000 description 9
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 238000000855 fermentation Methods 0.000 description 8
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- SAHIZENKTPRYSN-UHFFFAOYSA-N [2-[3-(phenoxymethyl)phenoxy]-6-(trifluoromethyl)pyridin-4-yl]methanamine Chemical compound O(C1=CC=CC=C1)CC=1C=C(OC2=NC(=CC(=C2)CN)C(F)(F)F)C=CC=1 SAHIZENKTPRYSN-UHFFFAOYSA-N 0.000 description 6
- CGIGDMFJXJATDK-UHFFFAOYSA-N indomethacin Chemical compound CC1=C(CC(O)=O)C2=CC(OC)=CC=C2N1C(=O)C1=CC=C(Cl)C=C1 CGIGDMFJXJATDK-UHFFFAOYSA-N 0.000 description 6
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- 239000002024 ethyl acetate extract Substances 0.000 description 4
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- 239000001888 Peptone Substances 0.000 description 3
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- 241000894006 Bacteria Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 241001508802 Diaporthe Species 0.000 description 1
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
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- 108090000790 Enzymes Proteins 0.000 description 1
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- REAYFGLASQTHKB-UHFFFAOYSA-N [2-[3-(1H-pyrazol-4-yl)phenoxy]-6-(trifluoromethyl)pyridin-4-yl]methanamine Chemical compound N1N=CC(=C1)C=1C=C(OC2=NC(=CC(=C2)CN)C(F)(F)F)C=CC=1 REAYFGLASQTHKB-UHFFFAOYSA-N 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
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- 238000003810 ethyl acetate extraction Methods 0.000 description 1
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- 229940021182 non-steroidal anti-inflammatory drug Drugs 0.000 description 1
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- 231100000816 toxic dose Toxicity 0.000 description 1
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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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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
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Abstract
The invention discloses a kind of indene derivatives derived from endophytic fungi in Excoecaria agallocha and its applications in preparing anti-inflammatory drugs.The present invention paints endogenetic fungus from one plant of seaDiaporthe The isolated indene derivative in sp.SYSU-HQ3.The bacterial strain was preserved in Guangdong Province's Culture Collection on August 4th, 2017, and deposit number is GDMCC No:60217.Such noval chemical compound has the generation for the NO for significantly inhibiting LPS induction, IC50It is respectively as follows: 8.5mM(1), 5.9mM(2), 4.2mM(3), 4.2mM(4), with the clinical application potentiality of anti-inflammatory treatment, it can be used for preparing anti-inflammatory drug, application prospect is good.
Description
Technical field
The invention belongs to pharmaceutical compound technical fields.More particularly, to a kind of the novel of sea paint endogenetic fungus source
Compound belongs to indene derivative and its application in preparing anti-inflammatory drugs.
Background technique
Inflammation is body by exogenous antigenic stimulus, and the immune defense process that body is taken.The generation of inflammation exists
Local organization can generate a series of complicated variation, mainly be presented with red, swollen, hot, pain and dysfunction, while inflammation can cause
General reaction, including fever, drowsiness, muscle protein degradation acceleration etc..When body is invaded by pro-inflammatory cytokine, body can be activated
Interior monocyte, macrophage etc. synthesize and discharge a variety of endogenous bioactive substances, as prostaglandin, nitric oxide,
Vasoactive amines etc., causes vasopermeability to increase, inflammatory cell chemotactic, to induce the generation of inflammation.In addition, inflammation with permitted
More chronic diseases such as arthritis, osteoporosis, asthma, cancer etc. are closely related.
Macrophage RAW264.7 is a kind of important cell model in anti-inflammatory screening.When with exogenous antigen lipopolysaccharides
LPS stimulating expression of macrophage can generate a series of immune response, wherein macrophage can generate NO in the whole process2 -。
Griess reagent is a kind of NO that can be generated with macrophage2 -Interaction has the azo dyes compounds of pigment.It is logical
The power of the absorbance of microplate reader detection dyestuff is crossed to judge the level of generation NO, to utilize this model discrimination little molecules in inhibiting
Agent.
There are two main classes: non-steroidal anti-inflammatory drugs and steroidal anti-inflammatory drugs for current clinically common anti-inflammatory agent.Although they
There are certain clinical antiphlogistic effects, but long-term a large amount of uses can generate a series of adverse reactions and tolerance, as gastric mucosa damages
Wound, hepar damnification, kidney damage etc..For the tolerance and adverse reaction for solving drug, new anti-inflammatory drug and correlation are found
The drug of novel framework types becomes the hot spot of anti-inflammatory drug research field.
Natural products is the important sources of lead compound, and the natural products in marine fungi source has sustainable
Property, environmentally friendly, the features such as metabolite is rich and varied, be always the important source of drug screening.The metabolite of fungi
The various actives such as antitumor with extensive physiological activity, such as antibacterial, immunological regulation is anti-inflammatory, and enzyme inhibits.Currently, true from ocean
The hot spot that new medicine source molecule has become international and domestic research is found in bacterium.
Summary of the invention
The technical problem to be solved in the present invention is to provide the indene derivative in a new class of marine fungi source, the series is new
Compound can inhibit the generation that LPS stimulating expression of macrophage generates NO, plays significant anti-inflammatory effect, has in preparing anti-inflammatory drug
There are long-range market prospects.
The object of the present invention is to provide one plant of sea paint endogenetic fungus Diaporthe sp.SYSU-HQ3.
Another object of the present invention is to provide a kind of indene derivatives derived from endophytic fungi in Excoecaria agallocha.
Another object of the present invention is to provide the indene derivatives derived from endophytic fungi in Excoecaria agallocha in anti-inflammatory drug
Using.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
One plant of sea paint endogenetic fungus Diaporthe sp.SYSU-HQ3, it is micro- to be preserved in Guangdong Province on August 4th, 2017
Biological inoculum collection, deposit number are GDMCC No:60217.
A kind of indene derivatives derived from endophytic fungi in Excoecaria agallocha, shown in structural formula such as following formula (I):
Specifically, such compound has type in 4, as shown in structural formula 1-4:
In addition, the indene derivatives derived from endophytic fungi in Excoecaria agallocha is from the sea paint endogenetic fungus Diaporthe
Isolated in the fermentation liquid of sp.SYSU-HQ3, the specific method is as follows:
S1. fungi seed culture:
The paint endogenetic fungus Diaporthe sp.SYSU-HQ3 of sea described in claim 1 is inoculated with slant medium, is cultivated
Seed liquor；
S2. fungi fermentation culture:
Seed liquor is accessed in solid rice fermentation culture medium, stands 1~2 month in 25~35 DEG C；
S3. it slightly mentions: the methanol of the product after step S2 fermented and cultured being extracted 2~5 times, concentrated extracting solution, by acquisition
Concentrated extract is extracted with ethyl acetate, and obtains ethyl acetate extract；
S4. it isolates and purifies: ethyl acetate extract being chromatographed with silica gel normal-phase chromatography and is separated；With petroleum ether/acetic acid
Ethyl ester is eluted, and collects 10%~50% ethyl acetate/petroleum ether part, then with columns such as silica gel, gel and/or C-18 reverse phases
Chromatography technology is isolated and purified.
Wherein it is preferred to the formula of slant medium described in step S1 are as follows: by quality ratio, glucose 0.3%, yeast
Extract 0.1%, peptone 0.1%~0.5%, agar 1.5%~2.5%, sodium chloride 1.5%~4%, water supplies 100%.
It is highly preferred that the formula of slant medium described in step S1 are as follows: by quality ratio, glucose 0.3%, yeast extracts
Object 0.1%, peptone 0.5%, agar 2.5%, sodium chloride 3%, water 98%.
Preferably, the condition of culture described in step S1 is 28~35 DEG C of cultures 4~10 days.
It is cultivated 6 days it is highly preferred that the condition of culture described in step S1 is 30 DEG C.
Preferably, the formula of solid rice fermentation culture medium described in step S2 are as follows: by quality ratio, rice: seawater=1:1
~2.
It is highly preferred that the formula of solid rice fermentation culture medium described in step S2 are as follows: by quality ratio, rice: seawater=
1:1.
Preferably, 35 DEG C standing 2 months in step S2.
Preferably, the number that methanol described in step S3 extracts is 3 times.
Preferably, petrol ether/ethyl acetate refers to 50~80% ethyl acetate and 20~50% petroleum ethers in step S4
Mixture.
It is highly preferred that petrol ether/ethyl acetate refers to 70% ethyl acetate and 30% petroleum ether in step S4.
In addition pass through it is experimentally confirmed that the indene derivatives derived from endophytic fungi in Excoecaria agallocha, which has, significantly inhibits LPS induction
NO generation, with anti-inflammatory treatment clinical application potentiality, can be used for preparing anti-inflammatory drug.
Therefore, such indene derivatives derived from endophytic fungi in Excoecaria agallocha application in preparing anti-inflammatory drugs, should be in this hair
Within bright protection scope.
And such indene derivatives derived from endophytic fungi in Excoecaria agallocha is able to suppress the drug that LPS induction NO is generated in preparation
In application, also should be within protection scope of the present invention.
The invention has the following advantages:
The present invention isolated a kind of indene derivative from the paint endogenetic fungus Diaporthe sp.SYSU-HQ3 of sea, should
Class noval chemical compound has the generation for the NO for significantly inhibiting LPS induction, IC50It is respectively as follows: 8.5 μM (1), 5.9 μM (2), 4.2 μM
(3), 4.2 μM (4), the clinical application potentiality with anti-inflammatory treatment, can be used for preparing anti-inflammatory drug, application prospect is good.
Specific embodiment
Further illustrate the present invention below in conjunction with specific embodiment, but embodiment the present invention is not done it is any type of
It limits.
Unless stated otherwise, the present invention uses reagent, method and apparatus for the art conventional reagent, method and are set
It is standby.
Unless stated otherwise, following embodiment agents useful for same and material are commercially available.
The acquisition of 1 sea paint endogenetic fungus Diaporthe sp.SYSU-HQ3 of embodiment
Fresh leaf of the inventor team from Guangdong Zhuhai waters mangrove plant sea paint Excoecaria agallocha L.
Isolated one plant of sea paint endogenetic fungus Diaporthe sp.SYSU-HQ3 in son, and Guangdong was preserved on August 4th, 2017
It saves Culture Collection (GDMCC), deposit number is GDMCC No:60217.
The separation of 2 compound of embodiment
The isolated novel indene derivative from the fermentation liquid of sea paint endogenetic fungus Diaporthe sp.SYSU-HQ3.
The specific method is as follows:
(1) the seed liquor culture of fungi Diaporthe sp.SYSU-HQ3: culture medium forms by weight are as follows: glucose
0.3%, yeast extract 0.1%, peptone 0.5%, agar 2.5%, sodium chloride 3%, water 98%；Test tube slant is made, chooses
Bacterial strain is taken to access inclined-plane, 30 DEG C are cultivated 6 days.
(2) fermented and cultured of fungi Diaporthe sp.SYSU-HQ3: solid rice fermentation culture medium: rice is utilized: sea
Water=1:1；Bacterial strain in seed is transferred in fermentation medium, stands 2 months in 35 DEG C of room temperature；
(3) methanol of the product after above-mentioned fermented and cultured is extracted 3 times, concentrated extracting solution uses the concentrated extract of acquisition
Ethyl acetate extraction, obtains ethyl acetate extract.
(4) ethyl acetate extract is chromatographed with silica gel normal-phase chromatography and is separated；It is washed with petrol ether/ethyl acetate
It is de-, collect 10%~50% ethyl acetate/petroleum ether part, then with column chromatography for separation technologies such as silica gel, gel, C-18 reverse phases,
It is isolated and purified and obtains compound 1-4.
The structural analysis of 3 compound of embodiment
Structured testing parsing is carried out to noval chemical compound 1-4, obtains following experimental data:
Noval chemical compound 1:C25H30O6, HRESI-MS:425.1961 [M-H]-(calculated value 425.1964).
Noval chemical compound 2:C25H30O6, HRESI-MS:425.1968 [M-H]-(calculated value 425.1964).
Noval chemical compound 3:C25H30O6, HRESI-MS:425.1964 [M-H]-(calculated value 425.1964).
Noval chemical compound 4:C25H30O6, HRESI-MS:425.1968 [M-H]-(calculated value 425.1964).
The NMR data of compound 1-4 is shown in Table 1, table 2.
NMR data (the CDCl of 1. compound 1 and 2 of table3, 500MHz/125MHz, ppm)
NMR data (the CDCl of 2. compound 3 and 4 of table3, 500MHz/125MHz, ppm)
The structural formula of compound 1-4 is as follows:
The anti-inflammatory cells screening model of 4 compound 1-4 of embodiment
1, the culture and processing of cell
264.7 cell of in vitro culture RAW, using the DMEM high glucose medium containing 10%FBS, in 37 DEG C, 5% titanium dioxide
Conventional maintenance culture and passage are carried out under the conditions of concentration of carbon.
2, compound intervention
(1) experimental method
Adjusting 264.7 cell density of RAW is 1 × 105The hole cells/ is simultaneously in logarithmic growth phase, and LPS (final concentration 1 is added
μ g/mL) inducing macrophage is in inflammatory conditions.
Compound 1-4 or Indomethacin are made into different drug concentrations using DMSO, each concentration sets 3 in parallel again
Hole, and Positive control wells (only plus LPS), negative control hole (cell and culture medium), blank control wells (culture medium) are set.
After culture 24 hours, 50 μ L of cell supernatant is taken to be added in 96 new orifice plates, the reagent I of NO detection kit is added
Inhibiting rate is calculated, and calculate IC using the content of Griess method measurement NO with each 50 μ L of II50。
Inhibiting rate=([NO2 -]LPS-[NO2 -]LPS+sample)/([NO2 -]LPS-[NO2 -]untreated)×100
(2) the results are shown in Table 3:
The effect that inhibits NO generate of the 3 compound 1-4 of table on RAW264.7 cellular level
The result shows that: indene analog derivative (1-4) shows the activity that stronger anti-NO is generated, IC50Less than respectively
8.5,5.9,4.2,4.2 μM, activity is than positive control Indomethacin IC50It is 37.5 μM about 4 times strong.
3, the influence of compound on intracellular vigor
MTT can be reduced to the blue crystallization of water-insoluble by the succinate dehydrogenase in living cells mitochondria, and be deposited on
In living cells, and dead cell has no this function.
(1) experimental method
264.7 cell of RAW of logarithmic growth phase, by 1 × 105The hole cells/ is inoculated in 96 orifice plates, the 100 every holes μ L.Add
Enter LPS (1 μ g/mL of final concentration) inducing macrophage and is in inflammatory conditions.
Compound 1-6 or Indomethacin are made into different drug concentrations using DMSO, each concentration sets 3 in parallel again
Hole, and Positive control wells (only plus LPS), negative control hole (cell and culture medium), blank control wells (culture medium) are set.
After culture 24 hours, every hole is added the 50 μ L of MTT solution of 1mg/mL and culture medium is sucked out after culture 4 hours, to
The DMSO of 150 μ L is added in every hole.Concussion shakes up.The absorbance value (A) at 490nm wavelength is measured with microplate reader.Drug is to thin
The inhibiting effect of intracellular growth indicates that survival rate is higher with survival rate, indicates that drug toxicity is lower.
Survival rate (%)=[(A1–A0)/(A2-A0)] × 100%, wherein A0The absorbance value of blank control, A1For sample
Absorbance value, A2For the absorbance value of Positive control wells.
The sample of 5 concentration is measured, dosage-inhibiting rate curve is drawn, obtains its CC50Value.Each sample replication three
It is secondary.
(2) the results are shown in Table 3
The result shows that: compound 1 does not show cytotoxic activity under 50 μM of concentration.And the cell of compound 2,3 and 4
Malicious CC50Respectively 20.5,29.2,27.3 μM, compared to the IC of itself50For, toxic concentration and half effective inhibition concentration
Range has a long way to go, and illustrates that it has very big safety.
Claims (3)
1. a kind of indene derivatives derived from endophytic fungi in Excoecaria agallocha, which is characterized in that shown in the following formula (I) of structural formula:
。
2. the application in preparing anti-inflammatory drugs of indene derivatives derived from endophytic fungi in Excoecaria agallocha described in claim 1.
3. indene derivatives derived from endophytic fungi in Excoecaria agallocha described in claim 1 is able to suppress what LPS induction NO was generated in preparation
Application in drug.
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| CN111154658B (en) * | 2020-01-13 | 2021-11-26 | 广州中医药大学(广州中医药研究院) | Marine fungus, novel skeleton heteroterpene derivative prepared from marine fungus, and preparation method and application of novel skeleton heteroterpene derivative |
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