CN102584680A - Aspochalasin U and preparing method and application thereof - Google Patents

Aspochalasin U and preparing method and application thereof Download PDF

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CN102584680A
CN102584680A CN2011104516932A CN201110451693A CN102584680A CN 102584680 A CN102584680 A CN 102584680A CN 2011104516932 A CN2011104516932 A CN 2011104516932A CN 201110451693 A CN201110451693 A CN 201110451693A CN 102584680 A CN102584680 A CN 102584680A
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aspochalasin
aspergillus
methanol
methyl alcohol
panel
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徐庆妍
刘军亮
胡志钰
黄耀坚
郑忠辉
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Xiamen University
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Xiamen University
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Abstract

Aspochalasin U and a preparing method and application thereof relate to a new antagonistic tumor necrosis factor-alpha (TNF-alpha) active compound. The molecular formula of the Aspochalasin U is C24H37NO5, and molecular weight of the Aspochalasin U is 419. The preparing method includes: fermenting aspergillus F00685 Aspergillus sp.F00685; inoculating the fermented aspergillus on a half seawater plate culture media (PDA) panel for culturing and activating in streaking mode, re-inoculating the fermented aspergillus on a fresh half seawater plate PDA panel after bacterial colony sends forth, and leading the half seawater plate PDA panel to serve as a seed panel after bacterial colony bestrews the whole panel and produces spores; and dividing the seed panel into 3mm*3mm small blocks, and inoculating the half seawater plate PDA fermented panel for culturing in block picking mode to obtain the Aspochalasin U. The Aspochalasin U compound is applied to preparation of anti-inflammatory drugs or lead compounds with other biological activities.

Description

Aspochalasin U
Technical field
The present invention relates to a kind of antagonism TNF-alpha active new compound, especially relate to a kind of separation purification method and application of a kind of novel compound of antagonism tumor necrosis factor-alpha.
Background technology
The expression of crossing of tumour necrosis factor-alpha (TNF-α) is to cause the present multiple disease that still can not cure fully clinically, like a major reason of rheumatoid disease property sacroiliitis, sieve's g grace disease, the necrosis of cerebral cell ischemia property etc.At present; The therapeutic modality of taking to this type disease is the progress that delays and stop disease, the pain that palliates a disease and cause; Comprise and adopt glucocorticosteroid (like retrocortine), NSAIDs (like cox 2 inhibitor) and immunosuppressor (general), but this type medicine all can not radical curing of disease like the Abbe west.The antibody class antagonist class medicine of developing to the protein properties of TNF-α; Like rhu TNFR:Fc (Etanercept), Ying Fuli former times (Infliximab), adalimumab (Adalimumab) etc.; The characteristic that can utilize Ag-Ab to mutually combine combines with the TNF-α of overexpression, plays the effect of antagonism TNF-α.But this type biological antagonist costs an arm and a leg, and the medical expense in 1 year needs 10,000 dollars approximately, and can only take the mode administration of injecting, and this brings inconvenience to the patient.The TNF-alpha inhibitor medicine of micromolecular taking orally; Having the clear and definite advantage of TNF-α biological antagonist action target spot, do not have the disadvantage of administration inconvenience again, is to demand the newtype drug developed at present urgently; But up to the present, still none comes out to the small-molecule drug that TNF-α crosses expression.
Chinese patent CN101485661 discloses a kind of Sinomenine derivate through antagonism TNF-signal path treatment autoimmune disease, and promptly suc as formula the purposes of the Sinomenine derivate of I, it can be used for preparing the compsn of prevention or treatment autoimmune disease.This invention also discloses the purposes of formula I compound in preparation tumor necrosis factor-alpha (TNF-α) signal path blocker compsn.
Summary of the invention
The object of the present invention is to provide a kind of Aspochalasin U.
Said compd A spochalasin U separates from the meta-bolites of aspergillus F00685 Aspergillus sp.F00685 on the PDA substratum; Said aspergillus F00685 Aspergillus sp.F00685 has been deposited in Chinese typical culture collection center on May 19th, 2011; Address: China, Wuhan, Wuhan University; Postcode 430072, preservation center deposit number is CCTCCNO:M2011179.
Said aspergillus F00685 Aspergillus sp.F00685 separates the stone solarization saltworks from east, Jinjiang, Fujian, can be described as fungal secondary meta-bolites Aspochalasin U.
The structure of said Aspochalasin U is: (3S, 3aR, 6S, 6aR, 12R, 13R; 151R, E)-6,12,13-trihydroxy-3-isobutyl-4,5,8-trimethyl-3; 3a, 6,6a, 9,10,11; 12,13,14-decahydro-1H-cycloundeca [d] isoindole-1,15 (2H)-dione, its structural formula is following:
Figure BDA0000126789220000021
The molecular formula of said Aspochalasin U is C 24H 37NO 5, molecular weight is 419; White powder is soluble in methyl alcohol, acetone and other organic solvent, is insoluble in low polar solvents such as chloroform.The new texture of the structure of this compound for not appearing in the newspapers as yet.On the cell model of TNF-alpha-2 antagonists screening, this compound shows medium antagonistic activity, and this compound is at the preparation anti-inflammatory medicaments or have in the lead matter of other bioactivity and use.
The preparation method of said Aspochalasin U is following:
1) aspergillus F00685 Aspergillus sp.F00685 is fermented;
In step 1), said fermentation can be adopted glucose-potato-nutrient agar, and the prescription of said glucose-potato-nutrient agar is glucose 20g, potato 200g, and agar 15g, half seawater is settled to 1L, 121 ℃ of sterilization 20min; Said potato can shred, and boils 30min, and 4 layers of filtered through gauze keep filtrating.
2) streak inoculation is on half seawater PDA flat board, cultivates activation, treats that bacterium colony grows the back renewed vaccination to half fresh seawater PDA flat board, treat bacterium colony be covered with whole flat board and produce spore after as seed plate;
In step 2) in, said cultivation activatory temperature can be 28 ℃.
3) the seed flat board is divided into the fritter of 3mm * 3mm, inoculates the dull and stereotyped back of half seawater solid PDA fermentation with the mode of choosing piece and cultivate, promptly get Aspochalasin U.
In step 3), said culture condition can place 28 ℃ of incubators to cultivate 14 days.
The separation purification method that below provides Aspochalasin U is following:
1) with above-mentioned steps 3) after the culture that obtained was cut into the about 0.5cm fritter of the length of side, with ETHYLE ACETATE, methyl alcohol and the lixiviate of Glacial acetic acid min. 99.5 mixing solutions, said ETHYLE ACETATE, methyl alcohol and Glacial acetic acid min. 99.5 were ETHYLE ACETATE by volume: methyl alcohol: Glacial acetic acid min. 99.5=80: 15: 5; But said lixiviate lixiviate 3~4 times, again filtrate decompression is concentrated into dried, extract medicinal extract;
2) extract medicinal extract is colourless with ETHYLE ACETATE aqueous solution extraction to water; Said ETHYLE ACETATE: the volume ratio of water can be 1: 1; Said concentrating under reduced pressure can adopt Rotary Evaporators to be evaporated to dried for 42 ℃; And then with methyl alcohol and petroleum ether extraction, said methyl alcohol: the volume ratio of sherwood oil can be 1: 1, and methyl alcohol is evaporated to dried fermented product extract mutually;
3) with fermented product extract with dissolve with methanol after middle hydraulic fluid phase reversed-phase silica gel column chromatography; Use pure water, 30% methanol-water, 50% methanol-water, 70% methanol-water, 100% methanol-water system wash-out respectively; Wherein the component of 50% methanol-eluted fractions is collected by every 200mL/ pipe, and the result's merging that detects according to thin-layer chromatography (TLC) contains the component of Aspochalasin U and is concentrated into dried;
Aspochalasin U is at silica gel G F 254By volume, chloroform: in the development system of methyl alcohol=10: 1, the Rf value is about 0.50 on the thin layer plate, under UV-irradiation He in the iodine cylinder, all can show, sulfuric acid soaks back high-temperature heating treatment TLC plate, and compound presents red-purple.
4) contain Aspochalasin U component again through the molecular sieve gel post (Sephadex LH-20,140g) chromatography, methanol-eluted fractions, flow velocity 15s/ drips, 3600s/ pipe is collected, and detects through TLC to merge the component that contains Aspochalasin U; (the acetone wash-out can obtain the pure article of Aspochalasin U to the target compound component for Sephadex LH-20,40g) chromatography through gel column once more.
Compd A spochalasin U provided by the invention when concentration is not higher than 100 μ g/mL, has the effect of antagonism TNF-α on L929 cell (l cell) model.
The present invention seeks small molecules TNF-alpha-2 antagonists from microbial metabolites, the active method separation and purification from the Aspergillus strain tunning of following the trail of of utilization obtains having the compd A spochalasin U of better antagonism TNF-α effect.
Description of drawings
Fig. 1 is the antagonistic action of Aspochalasin U to TNF-α.In Fig. 1, X-coordinate is Aspochalasin U concentration (μ g/mL), and ordinate zou is a survival rate.
Embodiment
Said aspergillus F00685 Aspergillus sp.F00685 separates the stone solarization saltworks from east, Jinjiang, Fujian, can be described as fungal secondary meta-bolites Aspochalasin U.Said compd A spochalasin U separates from the meta-bolites of aspergillus F00685 Aspergillus sp.F00685 on the PDA substratum.
The preparation method of said Aspochalasin U is following:
1) aspergillus F00685 Aspergillus sp.F00685 is fermented; Said fermentation can be adopted glucose-potato-nutrient agar, and the prescription of said glucose-potato-nutrient agar is glucose 20g, potato 200g, and agar 15g, half seawater is settled to 1L, 121 ℃ of sterilization 20min; Said potato can shred, and boils 30min, and 4 layers of filtered through gauze keep filtrating.
2) streak inoculation is on half seawater PDA flat board, cultivates activation for 28 ℃ again, treats that bacterium colony grows the back renewed vaccination to half fresh seawater PDA flat board, treat bacterium colony be covered with whole flat board and produce spore after as seed plate.
3) the seed flat board is divided into the fritter of 3mm * 3mm, inoculates half seawater solid PDA fermentation flat board with the mode of choosing piece and be placed on 28 ℃ of incubators and cultivated 14 days, promptly get Aspochalasin U.
The separation purification method that below provides Aspochalasin U is following:
1) with above-mentioned steps 3) after the culture that obtained was cut into the about 0.5cm fritter of the length of side, with ETHYLE ACETATE, methyl alcohol and the lixiviate of Glacial acetic acid min. 99.5 mixing solutions, said ETHYLE ACETATE, methyl alcohol and Glacial acetic acid min. 99.5 were ETHYLE ACETATE by volume: methyl alcohol: Glacial acetic acid min. 99.5=80: 15: 5; But said lixiviate lixiviate 3~4 times, again filtrate decompression is concentrated into dried, extract medicinal extract;
2) extract medicinal extract is colourless with ETHYLE ACETATE aqueous solution extraction to water; Said ETHYLE ACETATE: the volume ratio of water can be 1: 1; Said concentrating under reduced pressure can adopt Rotary Evaporators to be evaporated to dried for 42 ℃; And then with methyl alcohol and petroleum ether extraction, said methyl alcohol: the volume ratio of sherwood oil can be 1: 1, and methyl alcohol is evaporated to dried fermented product extract mutually;
3) with fermented product extract with dissolve with methanol after middle hydraulic fluid phase reversed-phase silica gel column chromatography; Use pure water, 30% methanol-water, 50% methanol-water, 70% methanol-water, 100% methanol-water system wash-out respectively; Wherein the component of 50% methanol-eluted fractions is collected by every 200mL/ pipe, and the result's merging that detects according to thin-layer chromatography (TLC) contains the component of Aspochalasin U and is concentrated into dried;
Aspochalasin U is at silica gel G F 254By volume, chloroform: in the development system of methyl alcohol=10: 1, the Rf value is about 0.50 on the thin layer plate, under UV-irradiation He in the iodine cylinder, all can show, sulfuric acid soaks back high-temperature heating treatment TLC plate, and compound presents red-purple.
4) contain Aspochalasin U component again through the molecular sieve gel post (Sephadex LH-20,140g) chromatography, methanol-eluted fractions, flow velocity 15s/ drips, 3600s/ pipe is collected, and detects through TLC to merge the component that contains Aspochalasin U; (the acetone wash-out can obtain the pure article of Aspochalasin U to the target compound component for Sephadex LH-20,40g) chromatography through gel column once more.
The structure of said Aspochalasin U is: (3S, 3aR, 6S, 6aR, 12R, 13R; 151R, E)-6,12,13-trihydroxy-3-isobutyl-4,5,8-trimethyl-3; 3a, 6,6a, 9,10,11; 12,13,14-decahydro-1H-cycloundeca [d] isoindole-1,15 (2H)-dione, its structural formula is following:
Figure BDA0000126789220000041
The molecular formula of said Aspochalasin U is C 24H 37NO 5, molecular weight is 419; White powder is soluble in methyl alcohol, acetone and other organic solvent, is insoluble in low polar solvents such as chloroform.The new texture of the structure of this compound for not appearing in the newspapers as yet.On the cell model of TNF-alpha-2 antagonists screening, this compound shows medium antagonistic activity, and this compound is at the preparation anti-inflammatory medicaments or have in the lead matter of other bioactivity and use.
Below provide the structure of cell model:
The L929 cell of logarithmic phase with trysinization after, be dispersed into 10 with the fresh DMEM culture medium culturing that contains 10%FBS (foetal calf serum, Gibco company) 6The density of individual/mL, every hole 100 μ L cell suspensions are added to 96 flat orifice plates, 37 ℃, 5%CO 2Incubator in overnight cultures.Sopped up substratum in second day; All be replaced by fresh serum-free DMEM substratum: the blank group does not contain cell; Have only 100 μ LDMEM substratum; Negative control group adds 98 μ L DMEM and 2 μ LDMSO, adds 98 μ L DMEM and 2 μ L TNF-α (final concentration of TNF-α is 2ng/mL) in the TNF-α group, and sample sets adds 96 μ L DMEM, 2 μ L TNF-α and the 2 μ L Aspochalasin L26 solution (sample is dissolved among the DMSO) of gradient dilution; Each concentration of each sample is all established three repetitions, contains 5%CO at 37 ℃ 2Incubator in cultivate 24h.Sop up whole substratum afterwards, be replaced by the fresh serum-free DMEM substratum of 90 μ L once more.Every hole adds 10 μ L CCK-8 solution (Japanese colleague chemical company), 37 ℃ of lucifuges reaction 2h, and ELIASA 450nm colorimetric estimation absorption value, the survival rate of cell is calculated in the back of averaging by following formula:
Figure BDA0000126789220000051
The effect of Aspochalasin U antagonism TNF-α on cell model is as shown in Figure 1, can find out, when Aspochalasin U concentration during less than 50 μ g/mL, the survival rate of cell increases with the increase of Aspochalasin U concentration.But this experiment shows Aspochalasin U antagonism TNF-α and crosses the necrocytosis that expression causes.

Claims (7)

1.Aspochalasin U; It is characterized in that it separates from the meta-bolites of aspergillus F00685 Aspergillus sp.F00685 on the PDA substratum; Said aspergillus F00685 Aspergillus sp.F00685 is deposited in Chinese typical culture collection center on May 19th, 2011, and preservation center deposit number is CCTCC NO:M2011179;
The structure of said Aspochalasin U is: (3S, 3aR, 6S, 6aR, 12R, 13R; 151R, E)-6,12,13-trihydroxy-3-isobutyl-4,5,8-trimethyl-3; 3a, 6,6a, 9,10,11; 12,13,14-decahydro-1H-cycloundeca [d] isoindole-1,15 (2H)-dione, its structural formula is following:
Figure FDA0000126789210000011
The molecular formula of said Aspochalasin U is C 24H 37NO 5, molecular weight is 419.
2. the preparation method of Aspochalasin U as claimed in claim 1 is characterized in that may further comprise the steps:
1) aspergillus F00685 Aspergillus sp.F00685 is fermented;
2) streak inoculation is on half seawater PDA flat board, cultivates activation, treats that bacterium colony grows the back renewed vaccination to half fresh seawater PDA flat board, treat bacterium colony be covered with whole flat board and produce spore after as seed plate;
3) the seed flat board is divided into the fritter of 3mm * 3mm, inoculates the dull and stereotyped back of half seawater solid PDA fermentation with the mode of choosing piece and cultivate, promptly get Aspochalasin U.
3. the preparation method of Aspochalasin U as claimed in claim 2 is characterized in that in step 1), and glucose-potato-nutrient agar is adopted in said fermentation; The prescription of said glucose-potato-nutrient agar is glucose 20g; Potato 200g, agar 15g, half seawater is settled to 1L.
4. the preparation method of Aspochalasin U as claimed in claim 2 is characterized in that in step 2) in, said cultivation activatory temperature is 28 ℃.
5. the preparation method of Aspochalasin U as claimed in claim 2 is characterized in that in step 3), and said culture condition places 28 ℃ of incubators to cultivate 14 days.
6. the separation purification method of Aspochalasin U as claimed in claim 1 is characterized in that may further comprise the steps:
1) after the culture that claim 2 step 3) is obtained is cut into the length of side and is the 0.5cm fritter; With ETHYLE ACETATE, methyl alcohol and the lixiviate of Glacial acetic acid min. 99.5 mixing solutions, said ETHYLE ACETATE, methyl alcohol and Glacial acetic acid min. 99.5 are ETHYLE ACETATE by volume: methyl alcohol: Glacial acetic acid min. 99.5=80: 15: 5; But said lixiviate lixiviate 3~4 times, again filtrate decompression is concentrated into dried, extract medicinal extract;
2) extract medicinal extract is colourless with ETHYLE ACETATE aqueous solution extraction to water; Said ETHYLE ACETATE: the volume ratio of water is 1: 1; Said concentrating under reduced pressure adopts Rotary Evaporators to be evaporated to dried for 42 ℃; And then with methyl alcohol and petroleum ether extraction, said methyl alcohol: the volume ratio of sherwood oil is 1: 1, and methyl alcohol is evaporated to dried fermented product extract mutually;
3) with fermented product extract with dissolve with methanol after middle hydraulic fluid phase reversed-phase silica gel column chromatography; Use pure water, 30% methanol-water, 50% methanol-water, 70% methanol-water, 100% methanol-water system wash-out respectively; Wherein the component of 50% methanol-eluted fractions is collected by every 200mL/ pipe, and the result's merging that detects according to thin-layer chromatography (TLC) contains the component of Aspochalasin U and is concentrated into dried;
4) contain Aspochalasin U component again through the molecular sieve gel column chromatography, methanol-eluted fractions, flow velocity 15s/ drips, and the 3600s/ pipe is collected, and detects through TLC to merge the component that contains Aspochalasin U; The target compound component is once more through gel filtration chromatography, and the acetone wash-out can obtain the pure article of Aspochalasin U.
7. Aspochalasin U is preparing anti-inflammatory medicaments or is having the application in the lead matter of other bioactivity according to claim 1.
CN2011104516932A 2011-12-29 2011-12-29 Aspochalasin U and preparing method and application thereof Pending CN102584680A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103787953A (en) * 2014-01-17 2014-05-14 杭州维康科技有限公司 Compound Aspochalasin V and preparation method and application thereof
CN107119087A (en) * 2017-03-29 2017-09-01 浙江大学 A kind of cytochalasins compound AspochalasinD preparation method and application

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103787953A (en) * 2014-01-17 2014-05-14 杭州维康科技有限公司 Compound Aspochalasin V and preparation method and application thereof
CN103787953B (en) * 2014-01-17 2015-12-02 杭州维康科技有限公司 Compd A spochalasin V and its preparation method and application
CN107119087A (en) * 2017-03-29 2017-09-01 浙江大学 A kind of cytochalasins compound AspochalasinD preparation method and application
CN107119087B (en) * 2017-03-29 2020-05-12 浙江大学 Preparation method and application of cytochalasin compound Aspochalasin D

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Application publication date: 20120718