CN104630078B - Fourth Ring aspergillus ketone compounds and its preparation method and application - Google Patents
Fourth Ring aspergillus ketone compounds and its preparation method and application Download PDFInfo
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Abstract
Fourth Ring aspergillus ketone compounds and its preparation method and application, are related to aspergillus ketone compounds.Fourth Ring aspergillus ketone compounds include Fourth Ring asperginon A, B, C and D.Culture medium flat plate is prepared, aspergillus is transferred on flat board and cultivates to obtain flat board strain, is inoculated in fluid nutrient medium and obtains seed liquor；It is transferred in fermentation medium and obtains fermentation culture medium, it is concentrated to dryness after extraction, filtered after methanol dissolving, filtrate is concentrated to dryness to obtain medicinal extract, it is colourless that ethyl acetate phase is extracted to again, collect ethyl acetate phase and be concentrated to dryness to obtain crude extract medicinal extract, it is mutually colourless that petroleum ether is extracted to again, collect methanol and be mutually concentrated into paste, dissolved with methanol, concentration crude extract medicinal extract is concentrated to give after filtering, anti-phase middle hydraulic fluid phase column chromatography is carried out, component 1 and 2 is eluted to obtain, component 1 obtains Fourth Ring asperginon A and B through twice chromatographic and a Semi-preparative high pressure liquid chromatogram；Component 2 obtains Fourth Ring asperginon C and D through once chromatographing with a Semi-preparative high pressure liquid chromatogram.
Description
Technical field
The present invention relates to aspergillus ketone compounds, more particularly, to Fourth Ring aspergillus ketone compounds and preparation method thereof and should
With.
Background technology
Inflammatory reaction is a part for organism immune response, is blood vessel or organizes to damage or the destructive stimulus such as pathogen
Local reaction.The cardinal symptom of inflammatory reaction has heating, pain, swelling, general red and body function forfeiture etc., to patient's
Quality of life has different degrees of influence.Inflammation causes rheumatic arthritis, IBD, neurodegenerative disorders, septicopyemia
Property the disease such as shock, atherosclerosis it is related[1], local chronic inflammatory, which reacts, can also induce body canceration[2]。
At present, the mechanism of action of anti-inflammatory drug is varied, such as blocks the key synthesized with the prostaglandin of inflammation-related
The effect of enzyme cyclooxygenase-2 (COX-2), or suppress inflammatory factor such as tumor necrosis factor-alpha, interleukins (IL), an oxygen
Change the induced expression of the toxicity molecules such as nitrogen (NO), free radical[3-5]Deng.Widely used anti-inflammatory agent is broadly divided into two classes：One kind is
NSAIDs (NSAIDs), such as Diclofenac, brufen, Indomethacin, naproxen；Another is COX-2 selection
Property inhibitor former times dry goods, such as celecoxib, etoricoxib[6].This two classes medicine all exists while slight illness is reduced inflammation
Stronger side effect, such as long-term taking NSAIDs can cause stomach rotten to the corn or ulcer and various angiocardiopathies[7], former times dry goods
Medicine all has an impact to coagulation function and renal function.
Natural products is secondary metabolite of the class from organisms such as animal, plant, microorganisms, because its complexity is more
The chemical constitution of sample and various bioactivity, occupy very important status in new drug development all the time.Ocean is micro-
Biological special living environment, possibility is provided for metabolism diversity, therefore is to produce the natural production that structure is novel, activity is various
The important sources of thing.The natural products of marine microorganism metabolism, to develop the anti-inflammatory agent that novel therapeutic effect is good, toxic side effect is low
Thing or other biological activity medicine provide valuable source.
Bibliography：
1.Maranhao,R.C.and A.C.A.Leite,Development of Anti-Atherosclerosis
Therapy Based on the Inflammatory and Proliferative Aspects of the Disease
[J].Curr.Pharm.Des.,2015.21(9):p.1196-1204.
2.Schwartsburd,P.M.,Chronic inflammation as inductor of pro-cancer
microenvironment:Pathogenesis of dysregulated feedback control[J].Cancer
Metastasis Rev.,2003.22(1):p.95-102.
3.Warner,T.D.,et al.,Nonsteroid drug selectivities for cyclo-
oxygenase-1rather than cyclo-oxygenase-2are associated with human
gastrointestinal toxicity:a full in vitro analysis[J]
.Proc.Natl.Acad.Sci.U.S.A.,1999.96(13):p.7563-7568.
4.Huang,X.-s.,et al.,Inhibitory effect of momordicin on inflammatory
factors generation by preventing nuclear translocation of nuclear factor-
kappa B[J].Zhongguo Dongmai Yinghua Zazhi,2013.21(7):p.583-588.
5.Matsui,T.,et al.,Hypothermia at 35℃ Reduces the Time-Dependent
Microglial Production of Pro-inflammatory and Anti-inflammatory Factors that
Mediate Neuronal Cell Death[J].Neurocrit.Care,2014.20(2):p.301-310.
6.Shaikh,S.R.and G.M.Nazeruddin,Multi component reactions and non‐
steroidal anti‐inflammatory drugs[J].J.Chem.Pharm.Res.,2014.6(12):p.505‐534,
30pp.
7.Al‐Turki,D.A.,et al.,Therapeutic and toxic effects of new NSAIDs
and related compounds:a review and prospective study[J].Int.J.Pharmacol.,
2010.6(6):p.813‐825.
The content of the invention
The first object of the present invention is to provide aspergillus (Aspergillus sp.) ZL01b-14.
The second object of the present invention is to provide Fourth Ring aspergillus ketone compounds and preparation method thereof.
The third object of the present invention, which is to provide Fourth Ring aspergillus ketone compounds, the application prepared in anti-inflammatory medicaments.
Aspergillus (Aspergillus sp.) ZL01b-14 is preserved in Chinese Typical Representative culture on December 26th, 2014
Thing collection, address：Wuhan, China Wuhan University, postcode：430072, collection deposit number is CCTCC NO：M
2014668。
The Fourth Ring aspergillus ketone compounds include four asperginon compounds, i.e. Fourth Ring asperginon A, Fourth Ring asperginon B,
Fourth Ring asperginon C and Fourth Ring asperginon D；
Fourth Ring asperginon A molecular formula is C22H26O9, molecular weight is 434.16, the absolute configuration of chiral carbon is 5aS, 6R,
6aR、10aR、11R、11aR；
Fourth Ring asperginon B molecular formula is C22H28O8, molecular weight is 420.18, the absolute configuration of chiral carbon is 5aS, 6R,
6aR、10aR、11R、11aR；
Fourth Ring asperginon C molecular formula is C21H24O9, molecular weight is 420.14, the absolute configuration of chiral carbon is 5aS, 6R,
6aR、10aR、11R、11aR；
Fourth Ring asperginon D molecular formula is C22H28O9, molecular weight is 436.46, the absolute configuration of chiral carbon is 5aS, 6S,
6aR、10aR、11R、11aS、12R；
Fourth Ring asperginon A, Fourth Ring asperginon B, Fourth Ring asperginon C and Fourth Ring asperginon D chemical structural formula it is as follows：
The preparation method of the Fourth Ring aspergillus ketone compounds, comprises the following steps：
1) Potato-dextrose-agar Half seawater medium flat board is prepared, by aspergillus (Aspergillus sp.)
ZL01b-14 is transferred on Potato-dextrose-agar Half seawater medium flat board, and flat board strain is cultivated to obtain in inversion；
2) by step 1) obtained flat board strain is inoculated in shaking table in the seawater fluid nutrient medium of Potato-dextrose half and trains
Support, obtain seed liquor；
3) by step 2) gained seed liquor be transferred in fermentation medium, be inverted fermentation after fermentation culture medium；
4) by step 3) obtained fermentation culture medium extracted with mixed organic solvents, and extract solution is concentrated under reduced pressure into dry, first
Filtered after alcohol dissolving, filtrate is concentrated under reduced pressure into dry medicinal extract again, it is colourless that medicinal extract is extracted to ethyl acetate phase with ethyl acetate and water,
Collect ethyl acetate phase and be concentrated under reduced pressure into dry crude extract medicinal extract, again with methanol is mutually colourless to petroleum ether with petroleum ether extraction, receipts
Collection methanol is mutually concentrated under reduced pressure into paste, is then dissolved, is concentrated under reduced pressure again after filtering with methanol, must concentrate crude extract medicinal extract；
5) by step 4) obtained concentration crude extract medicinal extract carries out anti-phase middle hydraulic fluid phase column chromatography, continuously terraced with methanol/water
Degree elutes to obtain component 1 and component 2, and it is bent that component 1 obtains Fourth Ring through gel filtration chromatography twice and a Semi-preparative high pressure liquid chromatogram
Mould ketone A and Fourth Ring asperginon B；Component 2 obtains Fourth Ring aspergillus through a gel filtration chromatography and a Semi-preparative high pressure liquid chromatogram
Ketone C and Fourth Ring asperginon D.
In step 1) in, the compound method of the Potato-dextrose-agar Half seawater medium can be：By peeling
Potato is cut into small pieces, and adds water and boils 20~40min, and filtered through gauze obtains potato filtrate, adds glucose, agar and Ban Hai
Water, adds water and is settled to 1000mL, and sterilising conditions are the 20min that sterilized at 121 DEG C；Glucose and potato in the potato filtrate
Mass ratio can be 1: the mass ratio of (10~20), the agar and potato can be 1: (15~20).
In step 2) in, the compound method of the seawater fluid nutrient medium of Potato-dextrose half can be：By the horse of peeling
Bell potato is cut into small pieces, and adds water and boils 20~40min, and filtered through gauze obtains potato filtrate, adds glucose and half seawater, and it is fixed to add water
Hold to 1000mL, sterilising conditions are the 20min that sterilized at 121 DEG C；Glucose and the mass ratio of potato in the potato filtrate
Can be 1: (10~20)；The condition of the shaking table culture can be 27~28 DEG C, 160~180rpm, and the time of culture is 4~6 days.
In step 3) in, the fermentation medium can use Potato-dextrose-agar -20%NaCl culture mediums, Ma Ling
The compound method of potato-glucose-agar -20%NaCl culture mediums can be：The potato of peeling is cut into small pieces, adds water and boils 20
~40min, filtered through gauze obtains potato filtrate, adds glucose, NaCl and agar, adds water and be settled to 1000mL, sterilising conditions
For the 20min that sterilized at 121 DEG C, the mass ratio of glucose and potato in the potato filtrate can be 1: (10~20), fine jade
Fat and the mass ratio of potato can be 1: (15~20), potato and NaCl mass ratio can be 1: (0.5~1.5).
In step 4) in, the mixed organic solvents can use ethyl acetate/methanol/formic acid, ethyl acetate, methanol, first
The volume ratio of acid can be 80: 15: 5；The volume ratio of the ethyl acetate and water can be 1: 1, and the volume ratio of methanol and petroleum ether can
For 1: 1；The temperature being concentrated under reduced pressure for each time can be 45 DEG C.
In step 5) in, the ratio change of the methanol/water continuous gradient elution can be 0/100 to 100/0 by volume；
The component 1 is through the gel filtration chromatography twice in gel filtration chromatography twice and a Semi-preparative high pressure liquid chromatogram
Sephadex LH-20 can be used, eluant, eluent is respectively methanol and acetone/methanol, the volume ratio of acetone and methanol can be 4: 1, one
Secondary half elution requirement for preparing high pressure liquid chromatography can be 60% methanol；
The component 2 can be adopted through the gel filtration chromatography in a gel filtration chromatography and a Semi-preparative high pressure liquid chromatogram
With Sephadex LH -20, eluant, eluent is acetone/methanol, and the volume ratio of acetone and methanol can be 4: 1, once half prepare high pressure liquid
The eluant, eluent of phase chromatogram can be 45% methanol.
Using MTS [3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromides, trade name tetrazolium bromide] method, inspection
The cytotoxicity of 4 prepared Fourth Ring aspergillins is surveyed, Fourth Ring asperginon A, B, C and D are suppressing bacteria lipopolysaccharide induction
Application in the reaction of RAW264.7 cellular inflammations.By detecting the change in concentration of cell anticusp inflammation factor, so as to distinguish that Fourth Ring is bent
Whether mould ketone A, B, C and D can suppress the reaction of RAW264.7 cellular inflammations.Test result indicates that：Fourth Ring asperginon can suppress thin
The inflammatory reaction of the lipopolysaccharide-induced RAW264.7 cells of bacterium.Fourth Ring asperginon is preparing treatment rheumatic arthritis, inflammatory
Have in the diseases associated with inflammation medicine that enteropathy, neurodegenerative disorders and septic shock etc. are mediated by proinflammatory inflammation factor extensive
Using.As can be seen here, the Fourth Ring aspergillus ketone compounds can be applied in anti-inflammatory medicaments are prepared, and the inflammation is included but not
It is limited to the inflammation that rheumatic arthritis, IBD, neurodegenerative disorders and septic shock etc. are mediated by proinflammatory inflammation factor
Disease.
The anti-inflammatory medicaments are also included using Fourth Ring asperginon A, B, C and D as active ingredient and containing conventional medicinal load
The pharmaceutical composition of body.
The present invention from one plant of marine alga grow nonparasitically upon another plant aspergillus altogether in the novel Fourth Ring aspergillus ketone chemical combination of isolated 4 structures
Thing, the anti-inflammatory experiment of cellular level shows that this four compounds have antiinflammatory action, can prepare treatment anti-inflammatory medicaments and resist
Applied in anti-inflammatory drugs composition, in addition, the Fourth Ring asperginon compound prepared by the present invention can also prepare bioactivity medicine
Applied in thing primer.
Brief description of the drawings
Fig. 1 is Fourth Ring asperginon A X-ray crystal diffraction structure charts.
Fig. 2 is inhibitions of the Fourth Ring asperginon A to inflammatory factor IL-6.
Fig. 3 is inhibitions of the Fourth Ring asperginon A to inflammatory factor IL-1 β.
Fig. 4 is inhibitions of the Fourth Ring asperginon D to inflammatory factor IL-6.
Fig. 5 is inhibitions of the Fourth Ring asperginon D to inflammatory factor IL-1 β.
Fig. 6 is inhibitions of the Fourth Ring asperginon B to NO.
Fig. 7 is inhibitions of the Fourth Ring asperginon B to inflammatory factor TNF-α.
Fig. 8 is inhibitions of the Fourth Ring asperginon C to NO.
Fig. 9 is inhibitions of the Fourth Ring asperginon C to inflammatory factor TNF-α.
Embodiment
Following examples will the present invention is further illustrated with reference to accompanying drawing.
The Fourth Ring aspergillus ketone compounds include four asperginon compounds, i.e. Fourth Ring asperginon A, Fourth Ring asperginon B,
Fourth Ring asperginon C and Fourth Ring asperginon D；
Fourth Ring asperginon A molecular formula is C22H26O9, molecular weight is 434.16, the absolute configuration of chiral carbon is 5aS, 6R,
6aR、10aR、11R、11aR；
Fourth Ring asperginon B molecular formula is C22H28O8, molecular weight is 420.18, the absolute configuration of chiral carbon is 5aS, 6R,
6aR、10aR、11R、11aR；
Fourth Ring asperginon C molecular formula is C21H24O9, molecular weight is 420.14, the absolute configuration of chiral carbon is 5aS, 6R,
6aR、10aR、11R、11aR；
Fourth Ring asperginon D molecular formula is C22H28O9, molecular weight is 436.46, the absolute configuration of chiral carbon is 5aS, 6S,
6aR、10aR、11R、11aS、12R；
Fourth Ring asperginon A, Fourth Ring asperginon B, Fourth Ring asperginon C and Fourth Ring asperginon D chemical structural formula it is as follows：
The preparation method of the Fourth Ring aspergillus ketone compounds, comprises the following steps：
1) Potato-dextrose-agar Half seawater medium flat board is prepared, by aspergillus (Aspergillus sp.)
ZL01b-14 is transferred on Potato-dextrose-agar Half seawater medium, and flat board strain is cultivated to obtain in inversion；The potato-
The compound method of glucose-agar Half seawater medium can be：The potato of peeling is cut into small pieces, add water boil 20~
40min, filtered through gauze obtains potato filtrate, adds glucose, agar and half seawater, adds water and be settled to 1000mL, sterilising conditions
For the 20min that sterilized at 121 DEG C；Glucose and the mass ratio of potato can be 1 in the potato filtrate: (10~20), it is described
Agar and the mass ratio of potato can be 1: (15~20).
2) by step 1) obtained flat board strain is inoculated in shaking table in the seawater fluid nutrient medium of Potato-dextrose half and trains
Support, obtain seed liquor；The compound method of the seawater fluid nutrient medium of Potato-dextrose half can be：The potato of peeling is cut
Into fritter, add water and boil 20~40min, filtered through gauze obtains potato filtrate, add glucose and half seawater, add water and be settled to
1000mL, sterilising conditions are the 20min that sterilized at 121 DEG C；Glucose and the mass ratio of potato can be 1 in the potato filtrate
: (10~20)；The condition of the shaking table culture can be 27~28 DEG C, 160~180rpm, and the time of culture is 4~6 days.
3) by step 2) gained seed liquor be transferred in fermentation medium, be inverted fermentation after fermentation culture medium；The hair
Ferment culture medium can use Potato-dextrose-agar -20%NaCl culture mediums, Potato-dextrose-agar -20%NaCl trainings
Supporting the compound method of base can be：The potato of peeling is cut into small pieces, adds water and boils 20~40min, filtered through gauze obtains potato
Filtrate, adds glucose, NaCl and agar, adds water and be settled to 1000mL, and sterilising conditions are the 20min, the horse of being sterilized at 121 DEG C
The mass ratio of glucose and potato in bell potato filtrate can be 1: the mass ratio of (10~20), agar and potato can be 1:
(15~20), potato and NaCl mass ratio can be 1: (0.5~1.5).
4) by step 3) obtained fermentation culture medium extracted with mixed organic solvents, and extract solution is concentrated under reduced pressure into dry, first
Filtered after alcohol dissolving, filtrate is concentrated under reduced pressure into dry medicinal extract again, it is colourless that medicinal extract is extracted to ethyl acetate phase with ethyl acetate and water,
Collect ethyl acetate phase and be concentrated under reduced pressure into dry crude extract medicinal extract, again with methanol is mutually colourless to petroleum ether with petroleum ether extraction, receipts
Collection methanol is mutually concentrated under reduced pressure into paste, is then dissolved, is concentrated under reduced pressure again after filtering with methanol, must concentrate crude extract medicinal extract；Institute
Ethyl acetate/methanol/formic acid can be used by stating mixed organic solvents, and ethyl acetate, methanol, the volume ratio of formic acid can be 80: 15: 5；
The volume ratio of the ethyl acetate and water can be 1: 1, and the volume ratio of methanol and petroleum ether can be 1: 1；The temperature being concentrated under reduced pressure for each time
Degree can be 45 DEG C.
5) by step 4) obtained concentration crude extract medicinal extract carries out anti-phase middle hydraulic fluid phase column chromatography, continuously terraced with methanol/water
Degree elutes to obtain component 1 and component 2, and it is bent that component 1 obtains Fourth Ring through gel filtration chromatography twice and a Semi-preparative high pressure liquid chromatogram
Mould ketone A and Fourth Ring asperginon B；Component 2 obtains Fourth Ring aspergillus through a gel filtration chromatography and a Semi-preparative high pressure liquid chromatogram
Ketone C and Fourth Ring asperginon D.The ratio change of the methanol/water continuous gradient elution can be 0/100 to 100/0 by volume；Institute
Stating component 1 can use through the gel filtration chromatography twice in gel filtration chromatography twice and a Semi-preparative high pressure liquid chromatogram
Sephadex LH-20, eluant, eluent is respectively methanol and acetone/methanol, and the volume ratio of acetone and methanol can be 4: 1, once half make
The elution requirement of standby high pressure liquid chromatography can be 60% methanol；The component 2 is through a gel filtration chromatography and once semi-preparative
Gel filtration chromatography in high pressure liquid chromatography can use Sephadex LH -20, and eluant, eluent is acetone/methanol, acetone and methanol
Volume ratio can be 4: 1, and once half eluant, eluent for preparing high pressure liquid chromatography can be 45% methanol.
Using MTS [3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromides, trade name tetrazolium bromide] method, inspection
The cytotoxicity of 4 prepared Fourth Ring aspergillins is surveyed, Fourth Ring asperginon A, B, C and D are suppressing bacteria lipopolysaccharide induction
Application in the reaction of RAW264.7 cellular inflammations.By detecting the change in concentration of cell anticusp inflammation factor, so as to distinguish that Fourth Ring is bent
Whether mould ketone A, B, C and D can suppress the reaction of RAW264.7 cellular inflammations.Test result indicates that：Fourth Ring asperginon can suppress thin
The inflammatory reaction of the lipopolysaccharide-induced RAW264.7 cells of bacterium.Fourth Ring asperginon is preparing treatment rheumatic arthritis, inflammatory
Have in the diseases associated with inflammation medicine that enteropathy, neurodegenerative disorders and septic shock etc. are mediated by proinflammatory inflammation factor extensive
Using.As can be seen here, the Fourth Ring aspergillus ketone compounds can be applied in anti-inflammatory medicaments are prepared, and the inflammation is included but not
It is limited to the inflammation that rheumatic arthritis, IBD, neurodegenerative disorders and septic shock etc. are mediated by proinflammatory inflammation factor
Disease.
The anti-inflammatory medicaments are also included using Fourth Ring asperginon A, B, C and D as active ingredient and containing conventional medicinal load
The pharmaceutical composition of body.
The invention provides the structure determination parameter of four novel compounds：
Fourth Ring asperginon A：White powder, high resolution mass spectrum (HR-FT-MS) measures m/z 457.1469 [M+Na]+, obtain
Molecular formula C22H26O9.Using deuterated methanol as solvent on 600MHz NMRs, internal standard is done with TMS (trimethyl silane),1H-
NMR and13C-NMR data are shown in Table 1.1H-NMR data are：δ 4.53 (1H, s), δ 3.31 (1H, d, 32.2), δ 2.83 (1H,
17.1), δ 2.76 (1H, d, 17.1), δ 2.30 (3H, s), δ 2.12 (1H, dq, 5.6,11.8), δ 1.99 (3H, s), δ 1.48
(3H, s), δ 1.36 (3H, s), δ 1.31 (3H, s), δ 1.25 (3H, d, 6.3).97.6 ° of [α] (c 0.25, MeOH)；UV
(MeOH):225nm and 314nm；IR(KBr):1733.83、1699.95、1539.66、1533.24、1436.61cm-1。
Fourth Ring asperginon B：White powder, high resolution mass spectrum (HR-FT-MS) measures m/z 421.1857 [M+H]+, obtain
Molecular formula C22H28O8.Using deuterated acetone as solvent on 600MHz NMRs, internal standard is done with TMS (trimethyl silane),1H-
NMR and13C-NMR data are shown in Table 1.1H-NMR data are：δ 5.72 (1H, s), δ 5.63 (1H, d, 4.7), δ 4.72 (1H, d,
2.3), δ 4.60 (1H, d, 3.9), δ 2.85 (1H, d, 2.6), δ 2.82 (1H, d, 2.6), δ 2.72 (1H, d, 16.7), δ 2.62
(1H, dd, 5.2,16.7), δ 2.19 (3H, s), δ 2.18 (1H, m), δ 2.01 (1H, dd, 12.4,16.4), δ 1.90 (1H, m), δ
1.89 (3H, s), δ 1.41 (3H, s), δ 1.32 (3H, s), δ 1.29 (3H, s), δ 1.08 (3H, d, 6.7).[α]67.4°(c
0.46, MeOH)；UV(MeOH):205nm and 290nm；IR(KBr)：1682.74th, 1570.56,1414.32 and 1382.90cm-1。
Fourth Ring asperginon C：White powder, high resolution mass spectrum (HR-FT-MS) measures m/z 421.1493 [M+H]+, obtain
Molecular formula C21H24O9.Using deuterated acetone as solvent on 600MHz NMRs, internal standard is done with TMS (trimethyl silane),1H-
NMR and13C-NMR data are shown in Table 1.1H-NMR data are：δ 6.02 (1H, s), δ 4.66 (1H, s), δ 3.33 (1H, d, 11.7), δ
2.84 (1H, d, 2.1), δ 2.75 (1H, s), δ 2.26 (3H, s), δ 2.15 (1H, m), δ 1.54 (3H, s), δ 1.32 (3H, s), δ
1.29 (3H, s), δ 1.28 (3H, d, 6.49).88 ° of [α] (c 0.1, MeOH)；UV(MeOH)：215nm、229nm、245nm、
275nm and 325nm；IR(KBr)：1734.07、1698.73、1539.67、1455.77、1222.63cm-1。
Fourth Ring asperginon D：White powder, high resolution mass spectrum (HR-FT-MS) measures m/z 459.1626 [M+Na]+, obtain
Molecular formula C22H28O9.Using deuterated acetone as solvent on 600MHz NMRs, internal standard is done with TMS (trimethyl silane),1H-
NMR and13C-NMR data are shown in Table 1.1H-NMR data are：δ 5.69 (1H, s), δ 5.66 (1H, d, 4.1), δ 4.71 (1H, d,
3.52), δ 4.67 (1H, d, 2.5), δ 4.54 (1H, d, 3.69), δ 3.97 (1H, bs), δ 2.88 (1H, dd, 2.51,16.9), δ
2.75 (1H, d, 16.9), δ 2.32 (1H, dq, 6.37,12.08), δ 2.22 (1H, d, 4.0), δ 2.21 (3H, s), δ 1.91 (3H,
S), δ 1.64 (3H, s), δ 1.32 (3H, s), δ 1.29 (3H, s), δ 1.21 (3H, d, 6.9).65.6 ° of [α] (c 0.25, MeOH)；
UV(MeOH)：220nm, 275nm and 299nm；IR(KBr)：1684.56、1569.02、1428.54、1257.90cm-1。
Table 1. Fourth Ring asperginon A, B, C and D's1H-NMR and13C-NMR data (data in ppm)
Specific embodiment given below.
1. culture medium prescription：
1) Potato-dextrose-agar Half seawater medium formula：200g peeling potatoes are taken, are cut into small pieces, adds water and boils
Boil in 30min, 8 layers of filtered through gauze, filtrate plus 20g glucose, 20g agar, 500mL seawater, running water is settled to 1000mL.
2) the seawater fluid nutrient medium of Potato-dextrose half：200g peeling potatoes are taken, are cut into small pieces, adds water and boils
Add 20g glucose in 30min, 8 layers of filtered through gauze, filtrate, 500mL seawater, running water is settled to 1000mL.
3) fermentation medium：200g peeling potatoes are taken, are cut into small pieces, adds water and boils 30min, 8 layers of filtered through gauze, filtrate
In plus 20g glucose and 20g agar, 200g NaCl add suitable quantity of water boil make its all dissolving after add potato filtrate in, originally
Water is settled to 1000mL.
2. zymotechnique：
By above-mentioned slant medium recipe configuration culture medium, test tube, 121 DEG C of sterilizing 20min, bevel culture are dispensed
Base.The aspergillus Aspergillus sp.ZL01b-14 of picking glycerol tube conservation are forwarded on slant medium, 28 DEG C of culture 4d；
By the recipe configuration culture medium of seed culture medium, sterilize, the slant strains of above-mentioned culture are transferred to seed culture medium by cooling
In, 28 DEG C, seed liquor is obtained after 180rpm shaking table cultures.Fermentation medium by recipe configuration it is good after, by above-mentioned cultured kind
Sub- liquid is transferred on fermentation medium, 28 DEG C of culture 30d.
3. Fourth Ring asperginon A, B, C and D's is refined：
Aspergillus (Aspergillus sp.) ZL01b-14 tunnings crude extract use in hydraulic fluid phase column chromatography, methanol/water from
0/100 to 100/0 (volume ratio) graded.Component 1 and component 2 are afforded when methanol/water ratio is 30/70.Component 1
Fourth Ring asperginon A is obtained through Sephadex LH-20 gel filtration chromatographies twice and a Semi-preparative high pressure liquid chromatogram and Fourth Ring is bent
Mould ketone B；Component 2 obtains Fourth Ring aspergillus through a Sephadex LH-20 gel filtration chromatography and a Semi-preparative high pressure liquid chromatogram
Ketone C and Fourth Ring asperginon D.The eluant, eluent of gel filtration chromatography twice of component 1 is respectively methanol and acetone：Methanol (4: 1, volume
Than), half elution requirement for preparing high pressure liquid chromatography is 60% methanol；The gel filtration chromatography of component 2 uses Sephadex
LH-20 eluant, eluents are acetone: methanol (4: 1, volume ratio), and half eluant, eluent for preparing high pressure liquid chromatography is 45% methanol.
4. Fourth Ring asperginon A recrystallization
Step 3 gained Fourth Ring asperginon A is all dissolved with methanol, takes appropriate lysate to be recrystallized, and picking is brilliant
Body carries out crystal X-Ray analyses and determined, and its absolute configuration is determined according to crystal data.
5. Fourth Ring asperginon A, B, C and D are tested the inhibitory activity of RAW264.7 cells：
By RAW264.7 cells (1 × 105Individual/mL) access in 96 well culture plates, per the μ L of hole 190, it is placed in 37 DEG C, 5%CO2
Incubator culture 12h, adds the μ L of Fourth Ring asperginon 10 of various concentrations, make its final concentration of 100,33.3,11.1,3.7 μM, often
Group is all provided with 3 repetitions, continues to cultivate 24h.20 μ L 5mg/mL MTT solution are added, continue to cultivate 4h.At ELIASA 570nm
The light absorption value in each hole is measured, wherein using DMSO as negative control, calculating inhibiting rate of the sample to cell.According to the cell of measurement
Inhibiting rate calculates compound effects in the 503nhibiting concentration (IC of cell using the softwares of GradPad Prism 550Value).
Cell inhibitory rate % (Inhibition%)=[1- (ODExperimental group-ODBlank group)/(ODNegative group-ODBlank group)] × 100%
Test result indicates that, the IC of Fourth Ring asperginon A, B, C and D to RAW264.7 cell growth inhibitions50Value is all higher than 100
μM, show that cell toxicant is not present to RAW264.7 cells in this series compound, can be neglected in the experiment for suppressing inflammatory factor
Slightly cytotoxic influence.
6. Fourth Ring asperginon A, B, C, D suppress the influence that LPS induction RAW264.7 cells discharge inflammatory factor
By detecting the change in concentration of cell anticusp inflammation factor, distinguish whether Fourth Ring aspergillin can suppress RAW264.7 thin
Born of the same parents discharge pro-inflammatory cytokine.Specific test method is as follows：
It is 5 × 10 to adjust concentration of cell suspension5/ mL, takes 1mL to be added in 24 orifice plates, puts 37 DEG C, 5%CO2Incubator culture,
Treat that 24h cell fusion degree reaches 80% or so, per hole add various concentrations Fourth Ring asperginon, make its final concentration of 20,40,80
μM (or 3.7,11.1,33.3,100 μM), DMSO is blank control, and every group is all provided with 3 repetitions.Continue to cultivate after 4h, add eventually
Concentration is 0.5 μ g/mL LPS, puts 37 DEG C and continues to cultivate 20h, takes 200 μ L of supernatant per hole, put -20 DEG C of preservations.
6.1ELISA methods survey cytokine TNF-α, IL-6, IL-1 β concentration
With reference to three kinds of inflammatory factor Mouse TNF-αs (BOSTER companies, Code#：EK0527)、Mouse IL-1β
(BOSTER companies, Code#：) and Mouse IL-6 (BOSTER companies, Cat# EK0394：EK0411) ELISA kit explanation
The concentration of three kinds of inflammatory factors in book, detection cell culture supernatant.Wherein it is with hydrocortisone (hydrocotisone)
Positive control, calculates inhibiting rate (inhibition) of the sample to inflammatory factor.
Inhibiting rate % (Inhibition%)=[1- (ODExperimental group-ODBlank group)/(ODLPS groups-ODBlank group)] × 100%
Influence of the 6.2 Fourth Ring asperginon compounds to RAW264.7 cell NO contents
The content (being operated according to specification) of nitric oxide (NO), nitric oxide inspection are detected by existing Griess methods
Test agent box is purchased to green skies biological reagent company, production code member：S0021.
6.3 experimental results show that the RAW264.7 cellular inflammation factors that Fourth Ring asperginon is induced bacteria lipopolysaccharide are shown
Different inhibitions, wherein Fourth Ring asperginon A and D have preferable inhibitory activity to IL-6 and IL-1 β (such as Fig. 2~Fig. 5 institutes
Show), and to NO and TNF-α when concentration is 40 μM without remarkable inhibiting activity (being less than 30%).；Fourth Ring asperginon B and C to NO and
TNF-α has a preferable inhibitory activity (as shown in figs. 6-9), and to IL-6 when concentration is 33.3 μM without significantly inhibiting work
Property (be less than 15%).
Test result indicates that：Fourth Ring asperginon can suppress the inflammatory reaction of the RAW264.7 cells of bacteria lipopolysaccharide induction.
Fourth Ring asperginon is preparing treatment rheumatic arthritis, IBD, neurodegenerative disorders and septic shock etc. by promoting
Had a wide range of applications in the diseases associated with inflammation medicine of inflammatory factor mediation.
Obtained Fourth Ring asperginon A is all dissolved with methanol, takes appropriate lysate to be recrystallized, and picking crystal enters
Row crystal X-Ray analyses are determined, and its absolute configuration is determined according to crystal data.Its spatial configuration figure is as shown in Figure 1.Recrystallization institute
It is methanol with solvent.
Claims (9)
1. Fourth Ring aspergillus ketone compounds, it is characterised in that bent including four asperginon compounds, i.e. Fourth Ring asperginon A, Fourth Ring
Mould ketone B, Fourth Ring asperginon C and Fourth Ring asperginon D；
Fourth Ring asperginon A molecular formula is C22H26O9, molecular weight is 434.16, the absolute configuration of chiral carbon is 5aS, 6R, 6aR,
10aR、11R、11aR；
Fourth Ring asperginon B molecular formula is C22H28O8, molecular weight is 420.18, the absolute configuration of chiral carbon is 5aS, 6R, 6aR,
10aR、11R、11aR；
Fourth Ring asperginon C molecular formula is C21H24O9, molecular weight is 420.14, the absolute configuration of chiral carbon is 5aS, 6R, 6aR,
10aR、11R、11aR；
Fourth Ring asperginon D molecular formula is C22H28O9, molecular weight is 436.46, the absolute configuration of chiral carbon is 5aS, 6S, 6aR,
10aR、11R、11aS、12R；
Fourth Ring asperginon A, Fourth Ring asperginon B, Fourth Ring asperginon C and Fourth Ring asperginon D chemical structural formula it is as follows：
2. the preparation method of aspergillus ketone compounds in Fourth Ring as claimed in claim 1, it is characterised in that comprise the following steps：
1) Potato-dextrose-agar Half seawater medium flat board is prepared, by aspergillus (Aspergillus sp.) ZL01b-14
It is transferred on Potato-dextrose-agar Half seawater medium flat board, flat board strain, the aspergillus are cultivated to obtain in inversion
(Aspergillus sp.) ZL01b-14 deposit number is CCTCC NO：M 2014668；
2) by step 1) obtained flat board strain is inoculated in shaking table culture in the seawater fluid nutrient medium of Potato-dextrose half, obtains
Seed liquor；
3) by step 2) gained seed liquor be transferred in fermentation medium, be inverted fermentation after fermentation culture medium；
4) by step 3) obtained fermentation culture medium extracts with mixed organic solvents, extract solution is concentrated under reduced pressure into dry, and methanol is molten
Filtered after solution, filtrate is concentrated under reduced pressure into dry medicinal extract again, it is colourless that medicinal extract is extracted to ethyl acetate phase with ethyl acetate and water, collects
Ethyl acetate phase is concentrated under reduced pressure into dry crude extract medicinal extract, and again with methanol is mutually colourless to petroleum ether with petroleum ether extraction, collects first
Alcohol phase is concentrated under reduced pressure into paste, is then dissolved with methanol, is concentrated under reduced pressure again after filtering, must concentrate crude extract medicinal extract；
5) by step 4) obtained concentration crude extract medicinal extract carries out anti-phase middle hydraulic fluid phase column chromatography, washed with methanol/water continuous gradient
Component 1 and component 2 are taken off to obtain, component 1 obtains Fourth Ring asperginon through gel filtration chromatography twice and a Semi-preparative high pressure liquid chromatogram
A and Fourth Ring asperginon B；Component 2 obtains Fourth Ring asperginon C through a gel filtration chromatography and a Semi-preparative high pressure liquid chromatogram
With Fourth Ring asperginon D.
3. the preparation method of aspergillus ketone compounds in Fourth Ring as claimed in claim 2, it is characterised in that in step 1) in, the horse
The compound method of bell potato-glucose-agar Half seawater medium is：The potato of peeling is cut into small pieces, add water boil 20~
40min, filtered through gauze obtains potato filtrate, adds glucose, agar and half seawater, adds water and be settled to 1000mL, sterilising conditions
For the 20min that sterilized at 121 DEG C；Glucose and the mass ratio of potato are 1 in the potato filtrate: (10~20), the fine jade
Fat and the mass ratio of potato are 1: (15~20).
4. the preparation method of aspergillus ketone compounds in Fourth Ring as claimed in claim 2, it is characterised in that in step 2) in, the horse
The compound method of the seawater fluid nutrient medium of bell potato-glucose half is：The potato of peeling is cut into small pieces, add water boil 20~
40min, filtered through gauze obtains potato filtrate, adds glucose and half seawater, adds water and be settled to 1000mL, sterilising conditions are 121
Sterilize 20min at DEG C；Glucose and the mass ratio of potato are 1 in the potato filtrate: (10~20)；The shaking table culture
Condition be 27~28 DEG C, 160~180rpm, time of culture is 4~6 days.
5. the preparation method of aspergillus ketone compounds in Fourth Ring as claimed in claim 2, it is characterised in that in step 3) in, the hair
Ferment culture medium uses Potato-dextrose-agar -20%NaCl culture mediums, Potato-dextrose-agar -20%NaCl cultures
The compound method of base is：The potato of peeling is cut into small pieces, adds water and boils 20~40min, filtered through gauze obtains potato filtrate,
Glucose, NaCl and agar are added, adds water and is settled to 1000mL, sterilising conditions are the 20min, the potato of being sterilized at 121 DEG C
The mass ratio of glucose and potato in filtrate is 1: the mass ratio of (10~20), agar and potato is 1: (15~20),
Potato and NaCl mass ratio are 1: (0.5~1.5).
6. the preparation method of aspergillus ketone compounds in Fourth Ring as claimed in claim 2, it is characterised in that in step 4) in, it is described mixed
Close organic solvent and use ethyl acetate/methanol/formic acid, ethyl acetate, methanol, the volume ratio of formic acid are 80: 15: 5；The acetic acid
The volume ratio of ethyl ester and water is 1: 1, and the volume ratio of methanol and petroleum ether is 1: 1；The temperature being concentrated under reduced pressure for each time is 45 DEG C.
7. the preparation method of aspergillus ketone compounds in Fourth Ring as claimed in claim 2, it is characterised in that in step 5) in, the first
The ratio change of alcohol/water continuous gradient elution is 0/100 to 100/0 by volume；
The component 1 is used through gel filtration chromatography twice and a Semi-preparative high pressure liquid chromatogram, twice gel filtration chromatography
Sephadex LH-20, eluant, eluent is respectively methanol and acetone/methanol, and the volume ratio of acetone and methanol is 4: 1, once half is prepared
The elution requirement of high pressure liquid chromatography is 60% methanol；
The component 2 is used through a gel filtration chromatography and a Semi-preparative high pressure liquid chromatogram, gel filtration chromatography
Sephadex LH -20, eluant, eluent is acetone/methanol, and the volume ratio of acetone and methanol is 4: 1, once half prepares high pressure liquid phase color
The eluant, eluent of spectrum is 45% methanol.
8. aspergillus ketone compounds in Fourth Ring as claimed in claim 1 are applied in anti-inflammatory medicaments are prepared；The inflammation includes wind
The inflammation that wet arthritis, IBD, neurodegenerative disorders and septic shock are mediated by proinflammatory inflammation factor.
9. application as claimed in claim 8, it is characterised in that the anti-inflammatory medicaments also include with Fourth Ring asperginon A, B, C and D
Pharmaceutical composition as active ingredient and containing conventional pharmaceutical carrier.
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| CN102187870A (en) * | 2010-03-19 | 2011-09-21 | 中国科学院烟台海岸带研究所 | Application of diterpene alkaloid compound serving as secondary metabolite of seaweed endophytic fungus |
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