CN107974412A - A kind of penicillium roqueforti for being used to prepare antiinflammatory active compound peniroquesine A and its application - Google Patents
A kind of penicillium roqueforti for being used to prepare antiinflammatory active compound peniroquesine A and its application Download PDFInfo
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- CN107974412A CN107974412A CN201711261581.4A CN201711261581A CN107974412A CN 107974412 A CN107974412 A CN 107974412A CN 201711261581 A CN201711261581 A CN 201711261581A CN 107974412 A CN107974412 A CN 107974412A
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- penicillium roqueforti
- peniroquesine
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/80—Penicillium
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
- A61K31/047—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates having two or more hydroxy groups, e.g. sorbitol
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- C—CHEMISTRY; METALLURGY
- 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
- C12P15/00—Preparation of compounds containing at least three condensed carbocyclic rings
Abstract
The present invention relates to microbial technology field, there is provided a kind of penicillium roqueforti (Penicillium roqueforti), deposit number are CGMCC No.14140.Penicillium roqueforti provided by the invention derives from yellow grass crow root, is the endogenetic fungus of yellow grass crow.Contain a kind of Dimeric sesquiterpene compound peniroquesine A with the novel new skeleton structure of 5/6,/5/,6/5 5 ring system in the metabolite of penicillium roqueforti of the present invention, there is significant anti-inflammatory activity.Dimeric sesquiterpene compound peniroquesine A can simply, efficiently be prepared using penicillium roqueforti provided by the invention, be used to prepare anti-inflammatory drug.Microbial fermentation, which prepares Dimeric sesquiterpene compound, has the advantages that the cycle is short, condition of culture is gentle, accessory substance is few, stereoselectivity is strong, cost is low.
Description
Technical field
The present invention relates to microbial technology field, more particularly to one kind is used to prepare antiinflammatory active compound
The penicillium roqueforti of peniroquesine A and its application.
Background technology
Endophyte of plant (endophyte) is to move in inside health plant tissue and organ in certain phase or whole stages
Fungi or bacterium, be prevalent in higher plant, woody, herbaceous plant, have in monocotyledon and dicotyledon
Endogenetic bacteria.Endophyte of plant be can in health plant tissue perch and do not cause substantive harm to plant, and and plant
The microorganism of harmonious symbiosis is established, a part of endophyte of plant can also benefit plant.
Hydro carbons and its oxygen-containing derivative of terpenoid (terpenes) --- the molecular formula for the multiple of isoprene unit
Thing, is a major class natural products with great diversity, including monoterpene, sequiterpene, diterpene, sesterterpene, triterpene etc..Perhaps
Polyterpene analog derivative has been currently being developed to the important drugs for the treatment of cancer, bacterium infection, malaria and other various human diseases, therefore
The synthesis of terpene just seems very important.But since biosynthetic pathway does not illustrate completely yet, terpenoid has non-mould
The factors such as the structure of block, no pervasive unified synthesis strategy, thus the available sources of terpenoid are dependent on natural
The extraction of product.
Dimeric sesquiterpene compound (sesterterpenes) largely belong in ocean various sponges and algae and
The secondary metabolite of its endogenetic fungus, extremely rare in terrestrial plant, conventional research finds the microorganism for having only a few
Such compound can be produced.According to the literature, Dimeric sesquiterpene compound is malicious, antitumor and anti-with anti-inflammatory, anti-cell
The multiple biological activities such as bacterium.
Due to extracting the of high cost of dimeric sesquiterpene compound, cycle length from marine organisms so that sesterterpenoids chemical combination
The anti-inflammatory activity of thing is difficult to be fully utilized, for further genralrlization dimeric sesquiterpene compound, it is necessary to find easier two times
Hemiterpene compound the way of production.
The content of the invention
It is used to prepare new Dimeric sesquiterpene compound peniroquesine A's it is an object of the invention to provide a kind of
Microorganism.
The present invention provides a kind of penicillium roqueforti (Penicillium roqueforti), deposit number CGMCC
No.14140。
The present invention also aims to provide penicillium roqueforti described in above-mentioned technical proposal to prepare antiinflammatory active compound
Application in peniroquesine A, the antiinflammatory active compound peniroquesine A have the structure shown in Formulas I：
Preferably, the penicillium roqueforti fermented and cultured is obtained into penicillium roqueforti tunning, Lou described in chromatographic isolation it is blue or green
Mould tunning, up to antiinflammatory active compound peniroquesine A.
Preferably, penicillium roqueforti fermentation fermentation medium used includes potato or rice.
It is described present invention also offers application of the penicillium roqueforti in anti-inflammatory drug is prepared described in a kind of preceding solution
The active ingredient of anti-inflammatory drug includes antiinflammatory active compound peniroquesine A and the one kind fermented by penicillium roqueforti
Or a variety of pharmaceutic adjuvants；
The antiinflammatory active compound peniroquesine A have the structure shown in Formulas I：
Preferably, in the anti-inflammatory drug of the preparation, antiinflammatory active compound peniroquesine A contents for 1~
99%.
Preferably, the formulation of the anti-inflammatory drug for tablet, capsule, solution, granule, pill, powder, paste,
Sublimed preparation, supensoid agent, pulvis, injection, suppository, creme, spray, patch, sustained release preparation, controlled release preparation or targeting preparation.
The present invention provides a kind of penicillium roqueforti (Penicillium roqueforti), deposit number CGMCC
No.14140.Penicillium roqueforti provided by the invention derives from yellow grass crow root, is the endogenetic fungus of yellow grass crow.Lou of the present invention
Contain a kind of sesterterpenoids with the new skeleton structure of novel [5,6,5,6,5] five ring system in the metabolite of ground mould
Compound peniroquesine A, there is significant anti-inflammatory activity, i.e., can be prepared one using penicillium roqueforti provided by the invention
The new Dimeric sesquiterpene compound of kind.
Using penicillium roqueforti provided by the invention can simply, efficiently prepare Dimeric sesquiterpene compound, microorganism hair
Ferment prepares Dimeric sesquiterpene compound with the cycle is short, condition of culture is gentle, accessory substance is few, stereoselectivity is strong, cost is low
Advantage；Mainly with the economic valency with higher for marine organisms extraction Dimeric sesquiterpene compound in compared with the prior art
Value, cost is low, easy to operate, is easy to mass produce, and new way is provided for the volume production of Dimeric sesquiterpene compound.
Penicillium roqueforti provided by the invention can be applied to prepare anti-inflammatory drug, and preparation includes antiinflammatory active compound
Peniroquesine A are the anti-inflammatory drug of active ingredient, and new selection is provided for exploitation anti-inflammatory drug.
Biological deposits explanation：
Penicillium roqueforti (Penicillium roqueforti), Chinese microorganism strain is deposited on July 20th, 2017
Preservation administration committee common micro-organisms center, address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and the Chinese Academy of Sciences is micro-
Biological study institute；Biological deposits numbering is CGMCC No.14140.
Brief description of the drawings
The HR-ESI-MS that Fig. 1 is antiinflammatory active compound peniroquesine A of the present invention is composed；
The HR-ESI-MS that Fig. 2 is antiinflammatory active compound peniroquesine A of the present invention composes testing result；
Fig. 3 is antiinflammatory active compound peniroquesine A in the present invention1H-NMR is composed；
Fig. 4 is antiinflammatory active compound peniroquesine A in the present invention13C-NMR and DEPT spectrums；
The HMBC spectrums that Fig. 5 is antiinflammatory active compound peniroquesine A in the present invention；
Fig. 6 is the hsqc spectrum of antiinflammatory active compound peniroquesine A in the present invention；
Fig. 7 is antiinflammatory active compound peniroquesine A in the present invention1H-1H COSY are composed.
Embodiment
The present invention provides a kind of penicillium roqueforti (Penicillium roqueforti), deposit number CGMCC
No.14140。
Penicillium roqueforti (Penicillium roqueforti) of the present invention is preserved in Chinese microorganism strain preservation
Administration committee's common micro-organisms center, deposit number are CGMCC NO.14140.The penicillium roqueforti is yellow from yellow grass crow
The endogenetic fungus of radix aconiti agrestis (Aconitum vilmorinianum Kom.), more particularly described penicillium roqueforti is from yellow grass crow root
Portion is isolated.
Specifically, the preparation method of penicillium roqueforti (Penicillium roqueforti) of the present invention is：
S1, take clean yellow grass crow root, soaks 0.5~5min with the liquor natrii hypochloritis of mass fraction 45~55%,
0.5~5min is carried out with the ethanol solution of quality volume fraction 75% again after cleaning, is cleaned, dry surface moisture；
S2, will be divided into long be no more than 5mm broken, by broken by the yellow grass crow root peeling that S1 steps are disinfected
It is inoculated in PDA culture medium, 26~30 DEG C of constant temperature incubations；
S3, every 20~30h observe 1 time, the pure bacterium colony of color phenotypes in PDA culture medium after inoculation is chosen, transfer
Rule and cultivate into new PDA culture medium, until obtaining single bacterium colony；
S4, by the isolated each single bacterium colony of S3 steps be inoculated in 24~27 DEG C of constant temperature incubations 5 in PDA culture medium respectively
~8d, choose colony edge be it is light yellow it is velvet-like, remaining be green, the back side is filemot bacterium colony, send to Kunming is large and holds up biology
Science and Technology Ltd. is accredited as penicillium roqueforti, up to penicillium roqueforti provided by the invention.
In the present invention, vaccination, which can be observed, in PDA culture medium, after culture 1d in the Loudi mould white point-like
Velvet-like mycelium；It can be observed to form the white fluffy body of round diameter 2cm or so, bacterium colony surface around vaccination after culture 3d
Relatively flat, reverse side is light yellow close to centre, and edge is colourless；Cultivate 5d back walls around inoculation dot into round diameter about
There are projection, bacterium colony center shape containing green particles in the bacterium colony of 4cm, bacterium colony surface, and edge is light yellow velvet-like, and the back side is yellowish-brown,
Edge is colourless；Colony edge is light yellow velvet-like after cultivating 7d, remaining is green, and the back side is yellowish-brown.
Present invention also offers penicillium roqueforti described in above-mentioned technical proposal to prepare antiinflammatory active compound
Application in peniroquesine A, the antiinflammatory active compound peniroquesine A have the structure shown in Formulas I：
Antiinflammatory active compound peniroquesine A shown in Formulas I are the generation of penicillium roqueforti described in above-mentioned technical proposal
Thank to product, be a kind of Dimeric sesquiterpene compound, there is a novel new skeleton structure of 5/6,/5/,6/5 5 ring system.
The antiinflammatory active compound peniroquesine A molecular formula are C25H40O2, anti-inflammatory activity as shown in Figure 1
HR-ESI-MS (high-resolution electrospray ionization mass spectrometry) spectrogram of compound peniroquesine A, 395.2923 [M+Na of m/z
]+.The present invention is by 1D/2D NMR (one-dimensional nuclear magnetic resonance wave spectrum and two dimensional NMR wave spectrum) and HR-ESI-MS to anti-inflammatory
Reactive compound peniroquesine A structures are identified,1H、13C, HMBC, HSQC and1H-1H COSY nuclear magnetic datas are shown in attached
Fig. 3~7, it may be determined that shown in formula I, its chemical name is its structure：
(1S,4R,8aS,10R,10aS,11bR)-1,6,6,8a,10a,11b-hexamethyl-2,3,3a,4,5,5a,
5b,5c,6,7,8,8a,9,10,10a,11b-hexadecahydro-1H-dicyclopenta[a,g]fluorene-4,10-
diol；
I.e.：(1S, 4R, 8aS, 10R, 10aS, 11bR) -1,6,6,8a, 10a, 11b- hexamethyl -2,3,3a, 4,5,5a,
5b, 5c, 6,7,8,8a, 9,10,10a, 11b- ten hexahydro -1H- bicyclic penta [a, g] fluorenes -4,10- glycol.
In the present invention, the method that the penicillium roqueforti prepares anti-inflammatory compound peniroquesine A, including following step
Suddenly：
(1) penicillium roqueforti is inoculated in fermentation medium and fermented, obtain penicillium roqueforti fermentate；
The deposit number of the penicillium roqueforti is CGMCC NO.14140；
(2) ultrasonic extraction after the penicillium roqueforti fermentate that the step (1) obtains is mixed with alcohol, obtains
Peniroquesine A crude extracts；
(3) peniroquesine A crude extracts are obtained into antiinflammatory active compound peniroquesine through chromatogram purification
A。
The penicillium roqueforti strain that deposit number is CGMCCNO.14140 is inoculated in fermentation by the present invention with 1% inoculum concentration
Ferment in culture medium, obtain penicillium roqueforti fermentate.The fermentation temperature is preferably 20~28 DEG C, more preferably 25~27 DEG C.
In the present invention, the fermentation time is 25~32d, is preferably 30d.The present invention connecing on fermentation medium to penicillium roqueforti
Kind amount is not particularly limited, and penicillium roqueforti strain described in picking accesses.
In the present invention, preferably potato or rice are included in the making raw material of the fermentation medium, more preferably
The fermentation medium made using potato as raw material.Fermentation medium of the present invention can make penicillium roqueforti growth and breeding i.e.
Can.
In the present invention, the fermentation method of the penicillium roqueforti is preferably solid fermentation, and penicillium roqueforti is in liquid fermentation
The metabolism time it is longer, the present invention can shorten fermentation time using solid fermentating mode.In the present invention, the solid fermentation
Temperature is 20~30 DEG C, is preferably 26~28 DEG C；The solid fermentation time is preferably 25~35d, is preferably 28~30d.
In the present invention, the fermentation process of the penicillium roqueforti is aerobic fermentation.
After obtaining the penicillium roqueforti fermentate, ultrasound carries after the present invention mixes penicillium roqueforti fermentate with alcoholic solution
Take, filter, the solvent in the filtrate removed, obtains peniroquesine A crude extracts.
In the present invention, the mass volume ratio of penicillium roqueforti fermentate and alcohol is (30~80) g:(50~150) mL, it is more excellent
Elect 40~60g as:80~120mL, is most preferably 50g:100mL.Alcohol of the present invention be preferably methanol, ethanol, propyl alcohol or
Isopropanol.The present invention uses alcohol to isolate anti-inflammatory compound peniroquesine A from penicillium roqueforti fermentate for solvent
Come.
In the present invention, the ultrasonic time is preferably 20~40min, more preferably 30min；The ultrasonic power is preferred
For 250W~350W, more preferably 300W.
The currently preferred solvent removed by the way of vacuum distillation in filtrate；Specifically, the present invention subtracts filtrate
Pressure is concentrated into no alcohol taste.The pressure being concentrated under reduced pressure is 10~15kPa, is preferably 13kPa；The temperature that is concentrated under reduced pressure is
48~55 DEG C, be preferably 50 DEG C.
After obtaining the peniroquesine A crude extracts, the present invention purifies peniroquesine A crude extracts, obtains
To antiinflammatory active compound peniroquesine A.Specifically, the present invention is with silica gel column chromatography elution peniroquesine A
Crude extract, eluent obtain antiinflammatory active compound peniroquesine A through chromatogram purification.Preferably, the present invention is using solidifying
Rubber column gel column chromatography purifies eluent.
It is currently preferred to be preferably using linear gradient elution method elution peniroquesine A crude extracts, the eluant, eluent
Volume ratio 100:0~20:1 chloroform-methanol.
Specifically, present invention chloroform-methanol dissolves peniroquesine A crude extracts, with
The silica gel mixing of 0.8~1.5 times of peniroquesine A crude extracts dry weight, removes solvent, fills column, use volume ratio successively
For 100:0、100:1、50:1、10:1、5:1 chloroform-methanol carries out gradient elution, respectively obtains volume ratio as 100:0、
100:1、50:1、10:1、5:1 chloroform-methanol elution fraction；It is 100 to take volume ratio:1 chloroform-methanol elution
Part, with volume ratio 200:1~80:1 petroleum ether:Acetone crosses silica gel column chromatography elution for solvent, obtains eluent.Compound
Peniroquesine A focus primarily upon the elution fraction of selection.
In the present invention, the method for removing solvent is distillation under vacuum；The temperature being concentrated under reduced pressure is 48~55
DEG C, it is preferably 50 DEG C.
In the present invention, the chloroform-ethanol solution for being used to dissolve peniroquesine A crude extracts is arbitrary volume
The eluant, eluent of ratio.It is currently preferred to be eluted using 200-300 mesh silica gel.
The present invention to obtained 100:It is described when 1 chloroform-methanol elution fraction carries out silica gel column chromatography elution
Eluting solvent flow velocity during elution is preferably 2~4mL/min, more preferably 3mL/min；The eluting solvent is preferably oil
Ether-acetone system.Specifically, when the present invention uses silica gel column chromatography to elution, petroleum ether is first used：Acetone (200:1) elute
300mL is to remove silica gel impurity, then uses petroleum ether：Acetone (120:1) elute 300mL with remove measure above principal point it is less miscellaneous
Matter, finally uses petroleum ether：Acetone (80:1) elution about 200mL target compounds are begun to flow out, and continue to use petroleum ether：Acetone (80:
1) rinse untill target compound disappears, the purer eluent containing target compound is merged.
After obtaining the eluent, invention carries out gel column chromatography purifying to the eluent, obtains anti-inflammatory compound
peniroquesine A.In the present invention, the gel column chromatography is preferably dextran gel column chromatography.The gel column color
Solvent during spectrum purifying is preferably methanol.
The solvent purified through gel column chromatography is preferably methanol；It is currently preferred to use sephadex column to compound
Purified.The methanol flow rate is preferably 0.4~0.6mL/min, more preferably 0.5mL/min.
Present invention also offers application of the penicillium roqueforti described in preceding solution in anti-inflammatory drug is prepared, the anti-inflammatory
The active ingredient of medicine includes antiinflammatory active compound peniroquesine A.Antiinflammatory active compound peniroquesine A
Structure and preparation method thereof do not repeating.
In the present invention, in the anti-inflammatory drug that the penicillium roqueforti is prepared preferably include mass percent 1~
99% anti-inflammatory compound peniroquesine A, more preferably 55~90%.It is of the present invention to contain anti-inflammatory compound
It can also include one or more pharmaceutic adjuvants in the anti-inflammatory drug of peniroquesine A, the excipient substance includes medicinal
One or more in carrier, diluent, adjuvant and excipient, can specifically include pharmaceutical preservative, antioxidant, fill out
Material, disintegrant, wetting agent, emulsifying agent, suspending agent, solvent, decentralized medium, coating, antiseptic or wait, which blend, absorbs delayer etc.；
The pharmaceutical carrier includes but not limited to liposome, alcohol plastid, polymer micelle, nano structured lipid carrier, solid lipid and receives
Meter Zai Ti, mesoporous silicon dioxide nano particle etc..
Piece is made using preparation method well known in the art in the medicine containing anti-inflammatory compound peniroquesine A
Agent, capsule, solution, granule, pill, powder, paste, sublimed preparation, supensoid agent, pulvis, injection, suppository, creme, spraying
Agent, patch, sustained release preparation, controlled release preparation or targeting preparation；Specifically, the tablet includes but not limited to sugar coated tablet, film
Garment piece agent, enteric coated tablet, buccal tablet etc.；The solution includes but not limited to injection, oral liquid；The capsule includes
But it is not limited to hard capsule, soft capsule etc..
In order to further illustrate the present invention, technical solution provided by the invention is retouched in detail with reference to embodiment
State, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1 separates penicillium roqueforti
Yellow grass crow root is cleaned to be placed in the liquor natrii hypochloritis of mass fraction 50% and soaks 1min, then with sterilizing
UP water afterwards rinses three times, then is placed in 75% ethanol solution and soaks 1min, and the same UP water with after sterilizing rinses after taking-up
Three times, the water on monkshood surface is blotted with filter paper.
The yellow grass crow root peeling that will be disinfected by above-mentioned, be cut into long broken no more than 5mm, by broken according to 5 pieces/
A to be inoculated on PDA plate, culture dish edge sealings are placed in 28 in GSP-9160MBE water isolation type constant incubators by sealed membrane
The hatching of DEG C constant temperature incubation.
In incubation every 24h observations once, the pure bacterium colony of color phenotypes is chosen and is placed in new PDA plate culture
Ware surface, line purifying repeats the above steps, untill obtaining the single bacterium colony of colonial morphology.By the single bacterium of colonial morphology
Strain is seeded to 25 DEG C of culture 7d on PDA slant mediums, observes colonial morphology.
Each single form bacterium colony that collection obtains is sent to Kunming Shuo Qing bio tech ltd and is identified, is identified
Saved backup to penicillium roqueforti at 4 DEG C.The penicillium roqueforti was deposited in Chinese microorganism strain preservation pipe on July 20th, 2017
Reason committee common micro-organisms center, address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and Chinese Academy of Sciences microorganism is ground
Study carefully institute；Biological deposits numbering is CGMCC No.14140.
The colony morphological observation of 2 penicillium roqueforti of embodiment
The penicillium roqueforti that deposit number is CGMCC NO.14140 is inoculated in PDA culture medium, 27 DEG C of constant temperature incubations, point
Not in 1d, 3d, 5d, 7d observation colonial morphology change.
Penicillium roqueforti is cultivated for 27 DEG C on PDA plate culture medium, can be observed have white point-like in vaccination front after 1d
Velvet-like mycelium；White fluffy body of the 3d back walls around vaccination into round diameter about 2cm is cultivated, bacterium colony surface is relatively flat,
Reverse side is light yellow close to centre, and edge is colourless；Cultivate bacterium of the 5d back walls around vaccination into round diameter about 4cm
Fall, there is a projection on bacterium colony surface, bacterium colony center shape containing green particles, and edge is light yellow velvet-like, and the back side is yellowish-brown, edge without
Color；Colony edge is light yellow velvet-like after 7d, remaining is green, and the back side is yellowish-brown.
Embodiment 3 prepares anti-inflammatory compound peniroquesine A
1st, actication of culture：Penicillium roqueforti (deposit number is CGMCC NO.14140) is inoculated on PDA slant mediums,
After 28 DEG C of 3~7d of constant temperature incubation, it is placed in 4 DEG C of refrigerators and stores for future use.
2nd, the preparation of fermentation medium：Clean potato is taken, the potato block for being divided into diameter 1cm is placed in tissue culture flasks,
50g/ bottles, high-temperature sterilization 30min, cooling, up to fermentation medium at 120 DEG C after tissue culture flasks capping.
3rd, the penicillium roqueforti that step 1 activates is inoculated into fermentation medium made from step 2 according to 1% inoculum concentration, added
After lid at 28 DEG C constant temperature incubation 30d, obtain penicillium roqueforti fermentate.
4th, the penicillium roqueforti fermentate that 50g steps 3 obtain is taken to be mixed with 100mL methanol according to mass volume ratio, mixing is molten
Liquid ultrasound 30min under the conditions of 40kHz, filtering, takes filtrate to be concentrated under reduced pressure into no alcohol taste, obtains 25.0g and contain
The crude extract of peniroquesine A.
5th, it is 2 with volume ratio:The peniroquesine A crude extracts of 1 chloroform-methanol 30mL dissolvings 25.0g, then
Mixed with 25.0g silica gel (200 mesh), be concentrated under reduced pressure and remove solvent, fill column；Successively with volume ratio 100:0,100:1,50:1,10:
1,5:1 chloroform:Methanol carries out gradient elution, merges each elution section and obtains 5 parts：Fr1~Fr5, takes wherein Fr2 parts
(100:1 chloroform；Methanol)；Petroleum ether is first used through silica gel column chromatography in Fr2 parts：Acetone (volume ratio 200:1) 300mL is eluted
To remove silica gel impurity, then use petroleum ether：Acetone (volume ratio 120:1) elute 300mL with remove measure above principal point it is less miscellaneous
Matter, finally uses petroleum ether：Acetone (volume ratio 80:1) elution about 200mL target compounds are begun to flow out, and continue to use petroleum ether：Third
Ketone (volume ratio 80:1) rinse untill target compound disappears, the purer eluent containing target compound is merged, is contained to obtain the final product
There is the eluent of peniroquesine A.Using methanol as solvent, dextran gel column chromatography purifying is carried out to eluent, it is dry,
Obtain 1.2g anti-inflammatory compound peniroquesine A.
The identification of 4 structure of embodiment
The anti-inflammatory compound peniroquesine A that Example 3 is prepared, by 1D/2D NMR, (one-dimensional nuclear-magnetism is total to
Vibration wave is composed and two dimensional NMR wave spectrum) and the obtained compound of HR-ESI-MS (high-resolution electrospray ionization mass spectrometry) identifications
Peniroquesine A and its structure.
(1) the HR-ESI-MS collection of illustrative plates of peniroquesine A is shown in attached drawing 1, and the result is shown in attached drawing 2：
With reference to the accompanying drawings 1 and attached drawing 2 shown in it can be seen that the molecular formula of the compound is C25H40O2(m/z:395.2923[M+
Na]+, calculated value：395.2926), degree of unsaturation 6.
(2) result of 1D/2DNMR detections is as shown in attached drawing 3~7.
The compound can be seen that by attached drawing 3,41H and13C NMR datas show that it contains a double bond (δH：5.57s,
1H；δC：135.5d), the compound is prompted to contain five rings.Its13C NMR show that the compound shares 25 carbon, including 6
CH3(δC：14.0q, 19.1q, 21.0q, 24.9q, 30.6q, 33.3q), 6 CH2(δC：26.0t,31.0t,40.9t,42.7t,
43.5t, 45.5t), 8 CH (δC：40.0d, 53.0d, 54.6d, 57.4d, 58.7d, 72.8d, 75.0d, 135.5d) and 5
Quaternary carbon (δC：43.7s,44.4s,48.3s,50.5s,147.9s).
The chemical potential of the H and connected C of compound peniroquesine A can be obtained by hsqc spectrum (Fig. 6)
Move δ ownership such as table 1：
1 compound peniroquesine A's of table1H NMR (600MHz) and13C NMR(150MHz)
By HMBC (Fig. 5) spectrum and1H-1CH in H COSY spectrums (Fig. 7)3-20(δH0.84s) with C-1 (δC40.0d), C-2
(δC31.0t) and C-9 (δCIt is 48.3s) related, CH3-21(δHIt is 0.95s) related to C-1's and C-9 to show CH3- 20 and CH3-
21 are connected with C-1 and C-9 respectively,1H-1CH in H COSY spectrums3- 20 and H-1 (δH1.84s), H-1 and H-2 (δH1.39, d, J=
13.6Hz；1.97, d, J=13.6Hz), H-2 and H-3 (δH1.89m), H-3 and H-4 (δH1.46m), H-4 and H-5 (δH
3.36m), H-5 and H-6 (δH0.91, d, J=10.4Hz；2.19, d, J=10.4Hz), H-6 and H-7 (δH2.40m), H-7 with
H-12 (δ H 1.61s), H-12 and H-13 (δH1.30, d, J=2.0Hz) correlation C is determined(20)-C(1)-C(2)-C(3)-C(4)-
C(5)-C(6)-C(7)-C(12)-C(13)Connecting framework.CH in HMBC spectrums3- 21 and C-4 (δCCorrelation 57.4d) determines C(4)-
C(9)The presence of key, CH3- 21 and C-8 (δCCorrelation 147.9s) determines C(8)-C(9)The connection of key, H-7 and C-8 and C-10
(δC135.5d) while there are HMBC correlations to show C-7 (δC53.0d) it is connected with double key carbon C-8, so far determines
The A rings and B rings of peniroquesine A.CH in HMBC spectrums3-22(δH1.13s) and C-10, C-11 (δC50.5s) and C-12
(δCCorrelation 54.6d) determines C(10)-C(11)-C(12)Structure junction fragment, be so far determined peniroquesine A's
C rings.CH in HMBC spectrums3-23(δH1.18s) and C-14 (δC43.7s) and C-13 (δCCorrelation 58.7d), determines C(13)-
C(14)The presence of key, CH3-24(δH0.98s) and CH3-25(δH0.92s) with C-19 (δC44.4s) and C-13 (δC 58.7d)
Correlation C is determined(13)-C(19)The connection of key.Equally, CH3- 24 and CH3- 25 and C-18 (δCHMBC 40.9t) is related,
CH3- 23 and C-17 (δCHMBC 42.7t) is related, H-17 (δH1.58, d, J=8.0Hz；1.70, m) with H-18 (δH
1.47m)1H-1H COSY are related, it is determined that C(19)-C(18)-C(17)-C(14)Connecting framework, so far determine
The F rings of peniroquesine A.CH3- 23 and C-15 (δCHMBC 45.5t) is related, CH3- 22 and C-16 (δC75.0d)
HMBC is related, and H-17 and H-181H-1H COSY are related, show that there are C(11)-C(16)-C(15)-C(14)Structure fragment,
So far the E rings of peniroquesine A are determined, so far also determine the structure of the compound.
In summary, it may be determined that the compound peniroquesine A structural formulas that embodiment 3 is prepared are：
The anti-inflammatory activity of 5 compound peniroquesine A of embodiment
Nitric oxide (nitric oxide, NO) has extensive and important biology adjusting function, inflammation, tumour and
Cardiovascular system etc. plays an important role.When immunocyte by microbial endotoxins, inflammatory mediator when stimulating, can generate big
The inducible nitric oxide synthase (induced NO synthase, iNOS) of amount, produces NO and carries out immune response, therefore presses down
NO generations processed are the direct indicators of compound anti-inflammatory activity.
(1) take the logarithm the mouse monokaryon macrophage RAW264.7 (being purchased from Chinese Academy of Sciences's Shanghai cell bank) in growth period, with 1 μ
G/mLLPS carries out induction stimulation, while adds the compound peniroquesine A processing that embodiment 3 is prepared, compound
The final concentration of peniroquesine A is followed successively by 3.125 μM, 6.25 μM, 12.5 μM, 25 μM and 50 μM five gradients, Mei Geti
Degree sets three Duplicate Samples, is cultivated under the conditions of 25 DEG C.
Set without medicine group and L-NMMA (total no inhibitor) positive drug group according to above-mentioned same steps at
Reason, as control.
(2) after cell pellet overnight (10h) culture, the NO growing amounts of each group are detected at 570nm；To the training being incubated overnight
MTS is added in nutrient solution and carries out cell survival rate detection, excludes the toxic effect of compound on intracellular.
NO generation inhibiting rates (%)=(be free of medicine group OD570nm- medicine group OD570nm)/be free of medicine group OD570nm×
100%；
IC50(half-inhibition concentration) is calculated by Reed＆Muench methods, obtains the NO suppressions of compound peniroquesine A
Make the IC of activity50For：20.69±1.61μM.
It is computed understanding, to the NO inhibitory activity of compound peniroquesine A, (positive control is：L-NMMA) sieve
In choosing experiment, generations of the compound peniroquesine A to nitric oxide (NO) shows obvious inhibitory activity, it half
Number inhibiting rate (IC50Value) be：20.69±1.61μM.And positive control L-NMMA is to the half inhibiting rate of nitric oxide (NO)
(IC50Value) be：32.88±2.59μM.Compound peniroquesine A are obvious to the generation inhibiting rate of nitric oxide (NO)
It is better than positive control L-NMMA.Therefore, peniroquesine A have the research valency as lead compound exploitation anti-inflammatory drug
Value.And a large amount of based on microbial fermentation and the method that simply produces anti-inflammatory compound peniroquesine A, not only can be with
Modern environment is protected and the demand of low-carbon economy, and further studying and opening for the later stage anti-inflammatory compound industrial volume production
Hair lays a solid foundation, and has significant application value.
Embodiment 6
1st, actication of culture：Penicillium roqueforti (deposit number is CGMCC NO.14140) is inoculated on PDA slant mediums,
After 28 DEG C of 3~7d of constant temperature incubation, it is placed in 4 DEG C of refrigerators and stores for future use.
2nd, the preparation of fermentation medium：50g rice in steep 12h in 40mL water is taken, rice is taken out and is placed in tissue culture flasks
In, 50g/ bottles, high-temperature sterilization 30min, cooling, up to fermentation medium at 120 DEG C after tissue culture flasks capping.
3rd, the penicillium roqueforti that step 1 activates is inoculated into fermentation medium made from step 2 according to 1% inoculum concentration, added
After lid at 26 DEG C constant temperature incubation 25d, obtain penicillium roqueforti fermentate.
4th, the penicillium roqueforti fermentate that 45g steps 3 obtain is taken to be mixed with 120mL methanol according to mass volume ratio, mixing is molten
Liquid ultrasound 30min under the conditions of 40kHz, filtering, takes filtrate to be concentrated under reduced pressure into no alcohol taste, obtains peniroquesine A
Crude extract.
5th, it is 2 with volume ratio:The peniroquesine A crude extracts of 1 chloroform-methanol 30mL dissolvings 25.0g, then
Mixed with 25.0g silica gel (300 mesh), be concentrated under reduced pressure and remove solvent, fill column；Respectively with volume ratio 100:0,100:1,50:1,10:
1,5:1 chloroform：Methanol carries out gradient elution, merges each elution section and obtains 5 parts：Fr1~Fr5, takes wherein Fr2 parts
(100:1 chloroform:Methanol)；Petroleum ether is first used through silica gel column chromatography in Fr2 parts：Acetone (volume ratio 200:1) 300mL is eluted
To remove silica gel impurity, then use petroleum ether：Acetone (volume ratio 120:1) elute 300mL with remove measure above principal point it is less miscellaneous
Matter, finally uses petroleum ether：Acetone (volume ratio 80:1) elution about 200mL target compounds are begun to flow out, and continue to use petroleum ether：Third
Ketone (volume ratio 80:1) rinse untill target compound disappears, the purer eluent containing target compound is merged, is contained to obtain the final product
There is the eluent of peniroquesine A.Using methanol as solvent, dextran gel column chromatography purifying is carried out to eluent, it is dry,
Obtain anti-inflammatory compound peniroquesine A.
The above is only the preferred embodiment of the present invention, not makees limitation in any form to the present invention.Should
Point out, for those skilled in the art, without departing from the principle of the present invention, if can also make
Dry improvements and modifications, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (7)
1. a kind of penicillium roqueforti (Penicillium roqueforti), deposit number is CGMCC No.14140.
2. application of the penicillium roqueforti described in claim 1 in antiinflammatory active compound peniroquesineA is prepared, its feature
It is, the antiinflammatory active compound peniroquesineA has the structure shown in Formulas I：
3. application according to claim 2, it is characterised in that the penicillium roqueforti fermented and cultured is obtained into penicillium roqueforti hair
Ferment product, penicillium roqueforti tunning described in chromatographic isolation, up to antiinflammatory active compound peniroquesineA.
4. application according to claim 3, it is characterised in that penicillium roqueforti fermentation fermentation medium used includes
Potato or rice.
5. application of the penicillium roqueforti described in claim 1 in anti-inflammatory drug is prepared, it is characterised in that the work of the anti-inflammatory drug
Property component include the antiinflammatory active compound peniroquesineA that is fermented by penicillium roqueforti and one or more medicinal auxiliary
Material.
The antiinflammatory active compound peniroquesineA has the structure shown in Formulas I：
6. application according to claim 4, it is characterised in that in the anti-inflammatory drug of the preparation, antiinflammatory active compound
PeniroquesineA contents are 1~99%.
7. application according to claim 4 or 5, it is characterised in that the formulation of the anti-inflammatory drug for tablet, capsule,
Solution, granule, pill, powder, paste, sublimed preparation, supensoid agent, pulvis, injection, suppository, creme, spray, patch, sustained release
Preparation, controlled release preparation or targeting preparation.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108558606A (en) * | 2018-06-05 | 2018-09-21 | 云南大学 | A kind of Dimeric sesquiterpene compound peniroquesines and its preparation method and application |
| CN109970538A (en) * | 2019-04-17 | 2019-07-05 | 中山大学 | The Dimeric sesquiterpene compound in a kind of marine fungi source and preparation method thereof and application in preparing anti-inflammatory drugs |
| CN115466268A (en) * | 2022-09-26 | 2022-12-13 | 云南大学 | Oxa-anthraquinone compound with antibacterial and anti-inflammatory activities, preparation method and application thereof, and pharmaceutical composition |
-
2017
- 2017-12-04 CN CN201711261581.4A patent/CN107974412B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| JIA-PENG WANG ET AL.: "Peniroquesines A−C: Sesterterpenoids Possessing a 5−6−5−6−5-Fused Pentacyclic Ring System from Penicillium roqueforti YJ-14", 《ORGANIC LETTERS》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108558606A (en) * | 2018-06-05 | 2018-09-21 | 云南大学 | A kind of Dimeric sesquiterpene compound peniroquesines and its preparation method and application |
| CN108558606B (en) * | 2018-06-05 | 2020-04-07 | 云南大学 | Sesterterpene compound peniroquesines, and preparation method and application thereof |
| CN109970538A (en) * | 2019-04-17 | 2019-07-05 | 中山大学 | The Dimeric sesquiterpene compound in a kind of marine fungi source and preparation method thereof and application in preparing anti-inflammatory drugs |
| CN109970538B (en) * | 2019-04-17 | 2022-04-05 | 中山大学 | Sesquiterpenoids derived from marine fungi, preparation method thereof and application thereof in preparing anti-inflammatory drugs |
| CN115466268A (en) * | 2022-09-26 | 2022-12-13 | 云南大学 | Oxa-anthraquinone compound with antibacterial and anti-inflammatory activities, preparation method and application thereof, and pharmaceutical composition |
| CN115466268B (en) * | 2022-09-26 | 2023-05-16 | 云南大学 | Oxaanthraquinone compound with antibacterial and anti-inflammatory activity, preparation method and application thereof, and pharmaceutical composition |
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