CN106399182B - Like grignard bacterium AUH-JLD49s and its application in the conversion of 3 '-demethyls-arctigenin - Google Patents
Like grignard bacterium AUH-JLD49s and its application in the conversion of 3 '-demethyls-arctigenin Download PDFInfo
- Publication number
- CN106399182B CN106399182B CN201610909771.1A CN201610909771A CN106399182B CN 106399182 B CN106399182 B CN 106399182B CN 201610909771 A CN201610909771 A CN 201610909771A CN 106399182 B CN106399182 B CN 106399182B
- Authority
- CN
- China
- Prior art keywords
- arctigenin
- jld49s
- demethyls
- strains
- conversion
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- 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
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- 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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
Abstract
The invention discloses a kind of love grignard bacterium AUH-JLD49s and its applications in the conversion of 3 '-demethyls-arctigenin, belong to bacteria technology field.Love grignard bacterium (EggerthellaSp.) AUH-JLD49s, deposit number are CGMCC No.13124.Its application includes the following steps: the culture of (1) strains A UH-JLD49s；(2) substrate 3 '-demethyl-arctigenin crude product and strains A UH-JLD49s are co-cultured and converted product isolates and purifies.Bacterial strain of the present invention can convert a kind of 3 '-demethyl of microorganism conversion product-arctigenin of arctigenin in Chinese medicine great burdock achene to 3 '-demethyls -4 '-and remove hydroxyl-arctigenin, it solves the problems, such as that 3 '-demethyls -4 '-remove hydroxyl-arctigenin resource shortage, there is great impetus to 3 '-DM-4 '-DH-AG pharmacological activity and new drug development.
Description
Technical field
The present invention relates to bacteria technology fields.
Background technique
Fructus arctii (Arctium lappaIt L. is) the biennial tubers medical and edible dual purpose plant of composite family, root and cauline leaf can be used as
Vegetables are edible, and root, fruit and seed can also be used as medicine.Great burdock achene is the dry mature fruit of fructus arctii, has anti-inflammatory, anticancer, resists
The multiple pharmacological effects such as HIV, anti-diabetic.Wherein arctiin (Arctiin) be Chinese medicine great burdock achene main active (Jilin Normal university's journal, 2011,11 (4): 64-65).Arctigenin (Arctigenin, abbreviation AG) is that arctiin sloughs grape
The free aglycone forms of sugar.Studies have shown that arctiin through sour water solution can efficiently produce arctigenin (Contemporary Chinese Chinese medicine,
2012,14 (6): 43-45).
For fructus arctii by after people or the intake in vivo of other animals, the main active arctiin in fructus arctii will be resided in enteron aisle
Microbial flora be converted into different products.It was reported according to Japanese scholars Mitsuhiko Nose etc. 1992, mouse intestinal bacterium
Group can convert substrate arctigenin to 3 '-demethyls-arctigenin (Planta Medicine, 1992, 58(6):520-
523)；2003, Masao Hattori research group co-cultured arctiin and human faecal mass, detected the metabolism of 6 kinds of arctiins
Product, metabolite 4 therein are that the product 3 '-demethyl -4 '-in the present invention removes hydroxyl-arctigenin.However, due to
Masao Hattori etc. using in human faecal mass microbial flora convert, metabolite 4 only in a certain specific time
It can detect and (co-culture 25-125 hours), the concentration of metabolite 4 is after co-cultivation reaches peak in about 75 hours or so
Decline rapidly again.It is well known that compared with single bacterial strain, unstable result when being converted using faecal bacterial community, changing effect
Poor reproducibility；In addition, metabolite type is more, and metabolism produces when Masao Hattori etc. is using people's faecal bacterial community conversion arctiin
The mesostates such as object 4 are only capable of just detecting in a certain special time period, be difficult to the centre including metabolite 4
Metabolite effectively prepared (Chemical and Pharmaceutical Bulletin, 2003, 51(4): 378-
384)；2007, Masao Hattori research group isolated one plant of Eubacterium bacterial strain from the fresh excrement sample of peopleEubacteriumsp. ARC-2, strains A RC-2 can convert substrate arctigenin to 7 kinds of different demethyl products
(Biological and Pharmaceutical Bulletin, 2007,30 (5): 904-911)；2013, this laboratory
Isolated one plant of Blaw spy's Pseudomonas bacterial strainBlautiaSp. AUH-JLD56, the bacterial strain can efficiently turn substrate arctigenin
Turn to a kind of product of 3 '-demethyls-arctigenin (Journal Agricultural Food Chemistry, 2013, 61
(49): 12060-12065).The method of strains A UH-JLD56 and its conversion preparation 3 '-demethyls-arctigenin was in 2013 years Shens
National inventing patent is reported, and was authorized to (ZL 201310368975.5) in 2015.The present invention is to have authorized patent of invention
Product 3 '-demethyl-arctigenin (ZL 201310368975.5) is substrate, isolated one plant of energy from the fresh excrement sample of people
The specific 3 '-demethyls -4 '-that are converted into of 3 '-demethyls-arctigenin are removed into hydroxyl-arctigenin (3 '-DM-4 '-DH-AG)
Bacterium bacterial strain, and using the bacterium bacterial strain as biological enzyme source, product 3 '-demethyl -4 '-is obtained by microbe transformation method and removes hydroxyl
Base-arctigenin.
To find the Structures of Natural Products analog with antiviral activity, German scholar Eich in 1996 etc. is with aryl two
Thiophene alkane is starting material, using a series of analogue for being chemically synthesized Lignanoids compounds, including the present invention
In product 3 '-DM-4 '-DH-AG.But compound 3 '-DM-4 '-DH-AG yield only 50% or so (Journal of Medicinal Chemistry, 1996,39,86-95).The catalyst Raney's nickel used in above-mentioned chemical synthesis is carcinogenic
Object, triphenylphosphine and butyl lithium are that so far, there is no any company using chemistry both at home and abroad with irritating risk catalyst
Method production or sale 3 '-DM-4 '-DH-AG.Due to compound 3 '-DM-4 '-DH-AG scarcity of resources, there are related compounds 3 '-
Report is few in terms of DM-4 '-DH-AG physiological activity.A current only document report, when 3 '-DM-4 '-DH-AG concentration is 10-8
Mol/L (i.e. 10-2 Micromoles per liter) when, product 3 '-DM-4 '-DH-AG can remarkably promote MCF-7 cell strainHJ2mm
Growth, and when concentration is increased to 10-7-10-5When mol/L, the product 3 '-DM-4 '-DH-AG then growth to cell strain MCF-7
Have no significant effect (Chemical and Pharmaceutical Bulletin, 2003,51 (4): 378-384).
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of love grignard bacterium (EggerthellaSp.) AUH-JLD49s and
Its application in the conversion of 3 '-demethyls-arctigenin, this bacterial strain can produce a kind of conversion of arctigenin in Chinese medicine great burdock achene
Object 3 '-demethyl-arctigenin (3 '-DMAG) is converted into 3 '-demethyls -4 '-and removes hydroxyl-arctigenin (3 '-DM-4 '-DH-
AG), 3 '-DM-4 '-DH-AG resource shortage of arctigenin converted product is solved the problems, such as, it is living to 3 '-DM-4 '-DH-AG pharmacology
Property and new drug development have great impetus.
In order to solve the above technical problems, the technical solution used in the present invention is:
A kind of love grignard bacterium AUH-JLD49s(EggerthellaSp.), deposit number is CGMCC No.13124.
Like grignard bacterium AUH-JLD49s(KX650621) it is that isolated one plant of Gram-negative from human excrement sample is stringent
Anaerobic bacteria bacterial strain, the bacterial strain can convert 3 '-for 3 '-demethyls-arctigenin (3 '-DMAG) in anaerobism work station and go first
Base -4 '-removes hydroxyl-arctigenin (3 '-DM-4 '-DH-AG).Related 3 '-DM-4 '-DH-AG microorganism biological route of synthesis is such as
Shown in lower:
Love grignard bacterium in the present inventionEggerthellaSp.AUH-JLD49s bacterial strain (abbreviation AUH-JLD49s) in
On October 19th, 2016 in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC) preservation, protects
Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 is hidden, deposit number is CGMCC No.13124.
Like grignard bacteriumEggerthellaThe taxology feature of sp.AUH-JLD49s bacterium are as follows:
1.0~2.0 millimeters of colony diameter, colony edge is neat, and there is protrusion at middle part, extends bacterium colony yellow with incubation time and adds
It is deep；Thallus is rod-shaped in BHI culture medium, and Gram's staining is negative.
It is including following the present invention also provides application of the strains A UH-JLD49s in the conversion of 3 '-demethyls-arctigenin
Step:
(1) culture of strains A UH-JLD49s；
(2) substrate 3 '-demethyl-arctigenin crude product and strains A UH-JLD49s are co-cultured, by substrate 3 '-demethyl-
Arctigenin crude product is converted into 3 '-demethyls -4 '-and removes hydroxyl-arctigenin；
(3) converted product isolates and purifies.
Wherein, the cultural method of strains A UH-JLD49s are as follows: the strains A UH-JLD49s for saving cryogenic freezing melts simultaneously
It is inoculated by the inoculum concentration of 15-20% volume ratio in the test tube for filling fresh BHI fluid nutrient medium, 37 DEG C in anaerobism work station
Lower culture 18-24 hours；The strains A UH-JLD49s in test tube is transferred to by Sheng with the inoculum concentration of 10-15% volume ratio again again
Have in fresh BHI fluid nutrient medium, continuation cultivates 18-24 hours as seed liquor in anaerobism work station.
Substrate 3 '-demethyl-arctigenin crude product and strains A UH-JLD49s co-culture method are as follows:
The seed liquor of strains A UH-JLD49s is transferred in BHI fluid nutrient medium by the inoculum concentration of 10-15% volume ratio and is trained
It supports, 3 '-demethyls-arctigenin crude product is then added, so that the concentration of 3 '-demethyls-arctigenin in the medium does not surpass
1.0 mM/ls are crossed, is cultivated 2-3 days in anaerobism work station, substrate 3 '-demethyl-arctigenin crude product can efficiently be turned
It turns to 3 '-demethyls -4 '-and removes hydroxyl-arctigenin (3 '-DM-4 '-DH-AG).
The mass percent of 3 '-demethyls-arctigenin and 3 '-demethyls-arctigenin crude product is not less than 90%.
The concentration of 3 '-demethyls-arctigenin is 100- in substrate 3 '-demethyl-arctigenin crude product of direct fermentation
200 mM/ls.
Converted product is isolated and purified using following methods:
Culture obtained in step (2) is extracted 2 times with isometric ethyl acetate, is evaporated, adds after extract liquor filtering
Enter 100% methanol solution, removes hydroxyl-arctigenin (3 '-with preparing column on HPLC and carrying out separation 3 '-demethyls -4 '-of preparation
DM-4 '-DH-AG).
Wherein, being separately cultured for strains A UH-JLD49s includes the following steps:
(1) acquisition and culture of human excrement sample
With sterilized cotton swab picking fresh excreta, it is put into 1 milliliter of fresh BHI fluid nutrient medium, sets anaerobism work station
It is cultivated 24 hours at a temperature of 37 DEG C inherent, the microbial flora as screening microorganisms with specific functions bacterial strain；
(2) it is separately cultured AUH-JLD49s
1. single colonie is separately cultured
The microbial flora cultivated 24 hours in anaerobism work station in advance is subjected to gradient with fresh BHI fluid nutrient medium
Dilution, being diluted to concentration respectively is 10–1, 10–2、10–3、10–4、10–5、10–6、10–7、10–8, then respectively by 100 microlitres of concentration point
It Wei 10–5、10–6、10–7、10–8Microbial flora dilution be evenly coated on the BHI solid medium being made ready beforehand for, will apply
There is the BHI solid medium of microbial flora dilution to be placed in anaerobism work station after culture 48 hours, is trained respectively from BHI solid
It supports tens of to the hundreds of single colonies of picking on base to set on BHI solid culture ware, and random number is carried out to the single colonie of institute's picking.
2. single colonie activity of conversion measures
Numbered single colonie is inoculated into respectively in the test tube for filling 1 milliliter of BHI fluid nutrient medium, 0.1 mmoles are added
That/liter 3 '-demethyls-arctigenin crude product, is cultivated 2 days in anaerobism work station, takes 100 microlitres of culture solutions and with 1000 microlitres
Ethyl acetate is extracted, and 100% methanol is added in extract liquor after being evaporated, detected with HPLC, and finally determining has conversion bottom
Object 3 '-demethyl-arctigenin function single colonie.
The preparation of substrate 3 '-demethyl-arctigenin crude product:
The patent of invention of authorization " the Blaw spy bacterium AUH- that the preparation of 3 '-demethyls-arctigenin is obtained with this laboratory
JLD56 and its application in arctigenin conversion " (ZL 201310368975.5).National inventing patent has been authorized with above-mentioned
Unlike, 3 '-demethyl of substrate-arctigenin used is to have authorized patent of invention (ZL in the present invention
201310368975.5) without the 3 '-demethyls-arctigenin crude product of purifying (culture i.e. obtained in step (2)) in.
Therefore, can save the purification procedures of the metabolite in patent of invention (ZL 201310368975.5), save the time and at
This.
The mass percent of 3 '-demethyls-arctigenin and 3 '-demethyls-arctigenin crude product is not less than 90%.
The present invention purifies obtained product 3 '-DM-4 '-DH-AG, and grinds for the first time to its Anticancer Activity in vitro
Study carefully.The result shows that concentration be the product 3 '-DM-4 '-DH-AG of 50-200 micromoles per liter to human colon cancer cell strain HCT116 and
The growth of Breast cancer lines MDA-MB-231 all has apparent inhibiting effect, and wherein concentration is 100 micromoles per liters 3 '-
DM-4 '-DH-AG is respectively 41.67% and 21.33% to the extracorporeal inhibiting rate of HCT116 and 231.With to product 3 '-DM-4 '-
DH-AG physiological function research deepens continuously, and from now on very likely develops 3 '-DM-4 '-DH-AG for new anticancer drug.
The beneficial effects of adopting the technical scheme are that
Strains A UH-JLD49s provided by the invention can remove a kind of converted product 3 '-of arctigenin in Chinese medicine great burdock achene
Methyl-arctigenin is converted into 3 '-demethyls -4 '-and removes hydroxyl-arctigenin, solves arctigenin converted product 3 '-and goes first
The problem of base -4 '-removes hydroxyl-arctigenin resource shortage, grinds arctigenin pharmacological active substance microbial metabolic products
Study carefully and new drug development has great impetus.
Detailed description of the invention
The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments；
Fig. 1 is the efficient liquid of 3 '-demethyl of strains A UH-JLD49s conversion of substrate-arctigenin in the embodiment of the present invention 1
Phase chromatogram；
Fig. 2 is the ultraviolet absorpting spectrum of 3 '-demethyls-arctigenin converted product；
Fig. 3 is the mass spectrogram of 3 '-demethyls-arctigenin converted product；
Fig. 4 is anaerobism bacterial strain AUH-JLD49s to substrate 3 '-demethyl-arctigenin conversion dynamic curve diagram；
Fig. 5 is that anaerobism bacterial strain AUH-JLD49s compares 3 '-demethyl of various concentration substrate-arctigenin conversion capability
Figure；
Fig. 6 is that product 3 '-demethyl -4 '-goes hydroxyl-arctigenin to human colon cancer cell strain HCT116 and human breast carcinoma
The inhibiting rate figure of cell strain MDA-MB-231.
Specific embodiment
Embodiment 1
1. strains A UH-JLD49s's is separately cultured
(1) acquisition and culture of human excrement sample
It with sterilized cotton swab picking people's fresh excreta, is put into 1 milliliter of fresh BHI fluid nutrient medium, sets anaerobism work
It stands and cultivate 24 hours at a temperature of inherent 37 DEG C, as the microbial flora for screening microorganisms with specific functions bacterial strain.
(2) it is separately cultured strains A UH-JLD49s
1. single colonie is separately cultured
The microbial flora cultivated 24 hours in anaerobism work station in advance is subjected to gradient with fresh BHI fluid nutrient medium
Dilution, being diluted to concentration respectively is 10–1, 10–2、10–3、10–4、10–5、10–6、10–7、10–8, then respectively by 100 microlitres of concentration point
It Wei 10–5、10–6、10–7、10–8Microbial flora dilution be evenly coated on the BHI solid medium being made ready beforehand for, will apply
There is the BHI solid medium of microbial flora dilution to be placed in anaerobism work station after culture 48 hours, is trained respectively from BHI solid
It supports tens of to the hundreds of single colonies of picking on base to set on BHI solid culture ware, and random number is carried out to the single colonie of institute's picking.
2. single colonie activity of conversion measures
Single colonie numbered, that culture is on BHI solid medium is inoculated into and fills 1 milliliter of BHI fluid nutrient medium
Test tube in, then be separately added into final concentration of 0.1 mM/l of 3 '-demethyls-arctigenin, cultivate 2 in anaerobism work station
It, takes 100 microlitres of culture solutions and is extracted with 1000 microlitres of ethyl acetate, and 100% methanol is added in extract liquor after being evaporated, and uses
HPLC detects the presence of product generation.Once culture has been detected product generation in certain test tube, purifying training just is carried out to bacterial strain
Feeding and culture presevation, while purifying and Structural Identification are carried out to product.
2. purifying culture, strain idenfication and the preservation of strains A UH-JLD49s
(1) the purifying culture of single colonie
The simple function microbial bacteria filtered out is fallen in into culture of crossing on BHI solid medium, after growing single colonie, then
It crosses, repeats at least 3 times or more, it is ensured that the single colonie form grown is consistent to the single colonie grown.By list after purification
For colony inoculation into 1 milliliter of BHI fluid nutrient medium, culture takes 100 microlitres of bacterium solution in 24 hours, is added to and fills 500 microlitres in advance
In the cryopreservation tube of sterilized skimmed milk power, add 2 millimeters thicks sterilized atoleine in advance on its surface after mixing, then by its
It is deposited in -80 DEG C of ultra low temperature freezer, rejuvenation culture is periodically carried out to the functional microorganism bacterial strain of low-temperature preservation and conversion is lived
Property measurement.
(2) strain idenfication is carried out to the microorganisms with specific functions bacterial strain isolated
Using simple function microbial strains thallus total DNA as template, with universal primer 27F/1492R(27F:5 '-
AGAGTTTGATCCTGGCTCAG-3′；1492R:5 '-GGTTACCTTGTTACGACTT-3 ') it is primer pair 16S rDNA sequence
PCR amplification is carried out, pcr amplification product delivers Shanghai Sheng Gong bioengineering Co., Ltd and carries out DNA sequencing, sequencing result warp
BLAST, which is compared, carries out similarity analysis, the 16S rDNA of the simple function microbial strains with other bacterial strains of GenBank database
Sequence and slowly love grignard bacteriumEggerthella lentaSimilitude be up to 99.8%, it is therefore, the conversion bacterial strain is tentatively true
It is set to love grignard Pseudomonas bacterial strain.It is AUH-JLD49s by the functional microorganism Strain Designation, and its 16S rDNA sequence is submitted
NCBI gene pool obtains bacterial strain number of registration (Accession Number) KX650621.
3. application of the strains A UH-JLD49s in the conversion of 3 '-demethyls-arctigenin
(1) culture of strains A UH-JLD49s
After people's enteron aisle isolated strains AUH-JLD49s freeze thawing of -80 DEG C of cryo-conservations, connect by the inoculum concentration of 20% volume ratio
Kind is into the test tube for filling fresh BHI fluid nutrient medium, bacterium solution in test tube after cultivating 18 hours at 37 DEG C in anaerobism work station
In uniform muddiness.Sheng is transferred to for muddy strains A UH-JLD49s has been grown in test tube with the inoculum concentration of 10% volume ratio again again
In the teat glass for having fresh BHI fluid nutrient medium, continuation cultivates 24 hours as seed liquor in anaerobism work station.
(2) substrate 3 '-demethyl-arctigenin crude product and strains A UH-JLD49s are co-cultured
By seed liquor the connecing by 10% volume ratio of above-mentioned cultured strains A UH-JLD49s in advance in anaerobism work station
Kind amount is transferred to the triangle culture in glassware for filling fluid nutrient medium, while the concentration that 3 '-demethyls-arctigenin is added is 150
MM/l 3 '-demethyls-arctigenin crude product so that 3 ' in culture medium-demethyl-arctigenin concentration be 1.0 mmoles
You/liter, it is cultivated 3 days in anaerobism work station, strains A UH-JLD49s can convert 3 '-DMAG of substrate to 3 '-DM-4 '-DH-
AG。
(3) with 3 '-demethyl of HPLC detection substrate-arctigenin by the conversion situation of strains A UH-JLD49s
It is taken out in 100 microlitres to 1.5 milliliters EP pipes of culture from above-mentioned triangular flask, 1000 microlitres of ethyl acetate is added to carry out
Extraction takes out 800 microlitres of upper layer ethyl acetate after standing centrifugation, 800 microlitre of 100% first is added after being evaporated in centrifuge concentrator
Alcoholic solution converts situation through HPLC detection substrate (see attached drawing 1).
(4) converted product isolates and purifies
If good through HPLC detection substrate conversion, culture in triangular flask is extracted 2 times with isometric ethyl acetate,
It is evaporated on a rotary evaporator after extract liquor filtering, 100% methanol solution is then added, cross the organic film that aperture is 0.45 micron
Separation preparation is carried out on HPLC with preparing column afterwards, collects metabolite 3 '-DM-4 '-DH-AG with clean triangular flask.
Embodiment 2
The isolated culture method of strains A UH-JLD49s is with embodiment 1, and strains A UH-JLD49s is in 3 '-demethyls-fructus arctii
Application in aglycon conversion includes the following steps:
(1) culture of strains A UH-JLD49s
After people's enteron aisle isolated strains AUH-JLD49s freeze thawing of -80 DEG C of cryo-conservations, connect by the inoculum concentration of 15% volume ratio
Kind is into the test tube for filling fresh BHI fluid nutrient medium, bacterium solution in test tube after cultivating 20 hours at 37 DEG C in anaerobism work station
In uniform muddiness.Sheng is transferred to for muddy strains A UH-JLD49s has been grown in test tube with the inoculum concentration of 15% volume ratio again again
In the teat glass for having fresh BHI fluid nutrient medium, continuation cultivates 18 hours as seed liquor in anaerobism work station.
(2) substrate 3 '-demethyl-arctigenin crude product and strains A UH-JLD49s are co-cultured
By seed liquor the connecing by 15% volume ratio of above-mentioned cultured strains A UH-JLD49s in advance in anaerobism work station
Kind amount is transferred to the triangle culture in glassware for filling fluid nutrient medium, while the concentration that 3 '-demethyls-arctigenin is added is 100
MM/l 3 '-demethyls-arctigenin crude product so that in culture medium concentration of substrate be 0.8 mM/l, in anaerobism work
Stand it is interior culture 2.5 days, strains A UH-JLD49s can convert 3 '-DMAG of substrate to 3 '-DM-4 '-DH-AG.
(3) 3 '-demethyl of HPLC detection substrate-arctigenin is by the conversion situation of strains A UH-JLD49s
It is taken out in 100 microlitres to 1.5 milliliters EP pipes of culture from above-mentioned triangular flask, 1000 microlitres of ethyl acetate is added to carry out
Extraction takes out 800 microlitres of upper layer ethyl acetate after standing centrifugation, 800 microlitre of 100% first is added after being evaporated in centrifuge concentrator
Alcoholic solution converts situation through HPLC detection substrate.
(4) converted product isolates and purifies
If good through HPLC detection substrate conversion, culture in triangular flask is extracted 2 times with isometric ethyl acetate,
It is evaporated on a rotary evaporator after extract liquor filtering, 100% methanol solution is then added, cross the organic film that aperture is 0.45 micron
Separation preparation is carried out on HPLC with preparing column afterwards, collects metabolite 3 '-DM-4 '-DH-AG with clean triangular flask.
Embodiment 3
The isolated culture method of strains A UH-JLD49s is with embodiment 1, and strains A UH-JLD49s is in 3 '-demethyls-fructus arctii
Application in aglycon conversion includes the following steps:
(1) culture of strains A UH-JLD49s
After people's enteron aisle isolated strains AUH-JLD49s freeze thawing of -80 DEG C of cryo-conservations, connect by the inoculum concentration of 18% volume ratio
Kind is into the test tube for filling fresh BHI fluid nutrient medium, bacterium solution in test tube after cultivating 24 hours at 37 DEG C in anaerobism work station
In uniform muddiness.Sheng is transferred to for muddy strains A UH-JLD49s has been grown in test tube with the inoculum concentration of 13% volume ratio again again
In the teat glass for having fresh BHI fluid nutrient medium, continuation cultivates 20 hours as seed liquor in anaerobism work station.
(2) substrate 3 '-demethyl-arctigenin crude product and strains A UH-JLD49s are co-cultured
By seed liquor the connecing by 12% volume ratio of above-mentioned cultured strains A UH-JLD49s in advance in anaerobism work station
Kind amount is transferred to the triangle culture in glassware for filling fluid nutrient medium, while the concentration that 3 '-demethyls-arctigenin is added is 200
MM/l 3 '-demethyls-arctigenin crude product so that in culture medium concentration of substrate be 0.6 mM/l, in anaerobism work
Stand it is interior culture 2 days, strains A UH-JLD49s can convert 3 '-DMAG of substrate to 3 '-DM-4 '-DH-AG.
(3) with 3 '-demethyl of HPLC detection substrate-arctigenin by the conversion situation of strains A UH-JLD49s
It is taken out in 100 microlitres to 1.5 milliliters EP pipes of culture from above-mentioned triangular flask, 1000 microlitres of ethyl acetate is added to carry out
Extraction takes out 800 microlitres of upper layer ethyl acetate after standing centrifugation, 800 microlitre of 100% first is added after being evaporated in centrifuge concentrator
Alcoholic solution converts situation through HPLC detection substrate.
(4) converted product isolates and purifies
If good through HPLC detection substrate conversion, culture in triangular flask is extracted 2 times with isometric ethyl acetate,
It is evaporated on a rotary evaporator after extract liquor filtering, 100% methanol solution is then added, cross the organic film that aperture is 0.45 micron
Separation preparation is carried out on HPLC with preparing column afterwards, collects metabolite 3 '-DM-4 '-DH-AG with clean triangular flask.
Structural Identification is carried out to the metabolite 3 '-DM-4 '-DH-AG of preparation, identification method and qualification result are as follows:
After the converted product peak of collection is evaporated, its purity is measured on analytic type high performance liquid chromatograph, then survey respectively
Determine ultraviolet absorption spectrum, mass spectrum, nuclear magnetic resonance spectroscopy and the carbon spectrum of metabolite.The result shows that the converted product is received 228 respectively
Rice and 278 nanometers have maximal ultraviolet absorption (see attached drawing 2), mass spectroscopy the result shows that, the metabolite molecular weight be 342
(see attached drawing 3), this just with 3 '-DM-4 '-DH-AG molecular formula C20H22O5It matches.According to the ultraviolet absorpting spectrum of product and divide
Son amount can be initially identified as 3 '-DM-4 '-DH-AG.For the chemical structure for further accurately parsing the metabolite, we
Determine respectively metabolite after purification nuclear magnetic resonance spectroscopy (1H-NMR) and carbon spectrum (13C-NMR).By spectrum elucidation, and
With the nuclear magnetic resonance spectroscopy and carbon of the 3 '-DM-4 '-DH-AG in document report spectrum compare and analyze (Chemical andPharmaceutical Bulletin, 2003,51 (4): 378-384), it is finally 3 '-by the metabolite precise Identification
DM-4′-DH-AG.Metabolite1H-NMR and13C-NMR result is as follows:
1H NMR (CDCl3, 400 MHz): δ 2.41-2.55 (2H, m, H-3, Ha-7′′), 2.59-2.62
(2H, m, H-2, Hb-7′′), 2.91 (1H, dd, J=13.90, 6.80 Hz, Ha-7′), 2.98 (1H, dd, J=
13.90, 5.30 Hz, Hb-7′), 3.82 (3H, s, -OCH3), 3.85 (3H, s, -OCH3), 3.90 (1H,
dd, J=9.18, 7.72 Hz, Ha-4), 4.14 (1H, dd, J=8.70, 7.12 Hz, Hb-4), 6.48 (1H, d,J=2.18 Hz, H-2′′), 6.51-6.58 (1H, dd, J=7.99, 1.94 Hz, H-6′′), 6.62 (1H, t, J
=2.18 Hz, H-2′), 6.68 (1H, br d, H-6′), 6.71 (1H, dd, H-4′), 6.82 (1H, d, J=
7.99 Hz, H-5′′), 7.14 (1H, t, J=7.72 Hz, H-5′)。
13C NMR (CDCl3, 100MHz): δ 179.1 (C-1), 156.1 (C-3′), 149.0 (C-3′′),
147.8 (C-4′′), 139.4 (C-1′), 130.5 (C-1′′), 129.8 (C-5′), 121.5 (C-6′), 120.7
(C-6′′), 116.2 (C-2′), 114.0 (C-4′), 111.8 (C-2′′), 111.4 (C-5′′), 71.5 (C-
4), -Me 55.9, -Me 55.8, 46.2 (C-2), 41.3 (C-3), 38.2 (C-7′′), 34.7 (C-7′)。
Strains A UH-JLD49s is analyzed to substrate 3 '-demethyl-arctigenin crude product conversion dynamic and conversion capability, and
Product 3 '-demethyl -4 '-goes hydroxyl-arctigenin to human colon cancer cell strain HCT116 and Breast cancer lines MDA-MB-
231 inhibiting effect:
1, strains A UH-JLD49s is to substrate 3 '-demethyl-arctigenin conversion dynamic
To understand 3 '-demethyl of strains A UH-JLD49s conversion of substrate-arctigenin speed, to determine optimal conversion
Time, we are determined 3 '-demethyl of strains A UH-JLD49s conversion of substrate-arctigenin conversion dynamic, specifically
Method is as follows:
Prior cultured seed liquor (cultural method is same as above) is inoculated by the inoculum concentration of 10% volume ratio and is filled
250 milliliters of triangle culture in glassware of 100 milliliters of liquid culture mediums, while the concentration that 3 '-demethyls-arctigenin is added is 150
MM/l 0.4 milliliter of 3 '-demethyls-arctigenin crude product, cultivated in anaerobism work station 0 hour, 3 hours, 6 hours,
100 microlitres of culture are taken after 9 hours, 12 hours, 15 hours, 18 hours, 21 hours, 24 hours respectively, with 1000 microlitres of second
Acetoacetic ester extraction takes 800 microlitres of extract liquor, and 100 microlitre of 100% methanol solution is added in extract liquor after being evaporated, and detects bottom with HPLC
Object is converted situation, draws bacterial strain according to different incubation time substrates and production concentration and converts dynamic (see figure 4), as shown in Figure 4,
Substrate has 84.7% or so product, 3 '-DM-4 '-DH-AG to be generated after being added 12 hours, after continuing culture 15 hours, is added
Substrate arctigenin there is 96.8% to be converted into 3 '-DM-4 '-DH-AG, product amount no longer rises substantially within the 24th hour.
2, strains A UH-JLD49s measures 3 '-demethyl of various concentration substrate-arctigenin conversion capability
Strains A UH-JLD49s is common in anaerobism work station with the 3 '-demethyls-arctigenin of various concentration respectively
Culture 3 days, then extracts culture with isometric ethyl acetate, and 100% methanol solution is added in extract liquor after being evaporated, and
Situation is converted with 3 '-demethyl of HPLC detection substrate-arctigenin crude product.The result shows that when substrate 3 '-demethyl-fructus arctii
When aglycon concentration is no more than 1.0 mM/ls, substrate 3 '-demethyl-arctiin of the strains A UH-JLD49s to various concentration
First average conversion (conversion ratio=production concentration/(remaining concentration of substrate+production concentration) × 100%) is 90% or more, such as bacterial strain
AUH-JLD49s is respectively 0.4 mM/l, 0.6 mM/l, 0.8 mM/l and 1.0 mM/ls to concentration
Average conversion is respectively 97.4%, 93.3%, 90.3% and 90.0%；When concentration of substrate increases to 1.2 mM/ls, bacterial strain
AUH-JLD49s decreases to the transformation efficiency of substrate mM/l, average conversion 84.7%；And work as bottom be added
When object concentration is 1.6 mM/ls, strains A UH-JLD49s sharply declines substrate mM/l conversion capability, average transformation
Rate is only that 18.0%(is shown in attached drawing 5).
3, product 3 '-demethyl -4 '-goes hydroxyl-arctigenin thin to human colon cancer cell strain HCT116 and human breast carcinoma
The inhibiting rate of born of the same parents' strain MDA-MB-231
Then each tumour cell of logarithmic growth phase uses RPMI-1640 culture medium with 0.25% trypsin digestion
Single cell suspension is made, adjustment cell concentration is 5 × 104A/milliliter, by cell suspension inoculation in 96 well culture plates, every hole
100 microlitres, each group is separately added into 50 micromoles per liters, 100 micromoles per liters, 200 micromoles per liters and 3 '-goes after cell is adherent
Methyl -4 '-goes hydroxyl-arctigenin and blank culture solution and positive control agent, respectively sets 3 multiple holes.Culture plate is placed in CO2Training
Support in case, the 20 microlitres/holes MTT are added in 37 DEG C of culture 24 hours backward each culture holes, after being incubated for 4 hours (suspension cell need to through from
The heart), careful inhale abandons supernatant culture solution, and 100 microlitres of DMSO are added in every hole, vibrate 10 minutes on oscillator, sufficiently dissolves first a ceremonial jade-ladle, used in libation
(Formazan) it crystallizes, microplate reader detects each hole absorbance A value, and measurement wavelength is 570 nanometers, calculates inhibiting rate IR=(1- medication
Group mean OD value/control group mean OD value) × 100%.The result shows that concentration is the 3 '-DM- of product of 50-200 micromoles per liter
4 '-DH-AG all have apparent suppression to the growth of human colon cancer cell strain HCT116 and Breast cancer lines MDA-MB-231
Production is used, and wherein concentration is that 100 micromoles per liter, 3 '-DM-4 '-DH-AG is respectively to the extracorporeal inhibiting rate of HCT116 and 231
41.67% and 21.33%(is shown in attached drawing 6).
Claims (7)
1. a kind of love grignard bacterium (Eggerthella sp.) AUH-JLD49s, deposit number is CGMCC No.13124.
2. application of the love grignard bacterium AUH-JLD49s in the conversion of 3 '-demethyls-arctigenin as described in claim 1,
It is characterized in that including the following steps:
(1) culture of strains A UH-JLD49s；
(2) substrate 3 '-demethyl-arctigenin crude product and strains A UH-JLD49s are co-cultured, by substrate 3 '-demethyl-fructus arctii
Aglycon crude product is converted into 3 '-demethyls -4 '-and removes hydroxyl-arctigenin；
(3) converted product isolates and purifies.
3. application of the love grignard bacterium AUH-JLD49s according to claim 2 in the conversion of 3 '-demethyls-arctigenin,
It is characterized by: step (1), the cultural method of strains A UH-JLD49s are as follows: the strains A UH-JLD49s for saving cryogenic freezing
Melt and be inoculated by the inoculum concentration of 15-20% volume ratio in the test tube for filling fresh BHI fluid nutrient medium, in anaerobism work station
It is cultivated 18-24 hours at interior 37 DEG C；The strains A UH-JLD49s in test tube is turned again with the inoculum concentration of 10-15% volume ratio again
It is connected to and fills in fresh BHI fluid nutrient medium, continuation cultivates 18-24 hours as seed liquor in anaerobism work station.
4. application of the love grignard bacterium AUH-JLD49s according to claim 3 in the conversion of 3 '-demethyls-arctigenin,
It is characterized by: step (2), substrate 3 '-demethyl-arctigenin crude product and strains A UH-JLD49s co-culture method are as follows: will
The seed liquor of strains A UH-JLD49s is transferred in BHI fluid nutrient medium by the inoculum concentration of 10-15% volume ratio and is cultivated, then plus
Enter 3 '-demethyls-arctigenin crude product, so that the concentration of 3 '-demethyls-arctigenin in the medium is no more than 1.0 mmoles
You/liter, it is cultivated 2~3 days in anaerobism work station, substrate 3 '-demethyl-arctigenin crude product Efficient Conversion is gone into first for 3 '-
Base -4 '-removes hydroxyl-arctigenin.
5. love grignard bacterium AUH-JLD49s according to claim 2 or 4 answering in the conversion of 3 '-demethyls-arctigenin
With, it is characterised in that: 3 '-demethyls-arctigenin and the mass percent of 3 '-demethyls-arctigenin crude product are not less than
90%.
6. application of the love grignard bacterium AUH-JLD49s according to claim 4 in the conversion of 3 '-demethyls-arctigenin,
It is characterized by: in 3 '-demethyls-arctigenin crude product 3 '-demethyls-arctigenin concentration be 100-200 mMs/
It rises.
7. application of the love grignard bacterium AUH-JLD49s according to claim 2 in the conversion of 3 '-demethyls-arctigenin,
It is characterized by: step (3), the isolation and purification method of converted product are as follows: will be obtained in step (2) with isometric ethyl acetate
Culture extract 2 times, extract liquor filtering after be evaporated, be added 100% methanol solution, separation system is carried out on HPLC with column is prepared
Standby 3 '-demethyls -4 '-remove hydroxyl-arctigenin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610909771.1A CN106399182B (en) | 2016-10-19 | 2016-10-19 | Like grignard bacterium AUH-JLD49s and its application in the conversion of 3 '-demethyls-arctigenin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610909771.1A CN106399182B (en) | 2016-10-19 | 2016-10-19 | Like grignard bacterium AUH-JLD49s and its application in the conversion of 3 '-demethyls-arctigenin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106399182A CN106399182A (en) | 2017-02-15 |
| CN106399182B true CN106399182B (en) | 2019-07-09 |
Family
ID=58013213
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610909771.1A Active CN106399182B (en) | 2016-10-19 | 2016-10-19 | Like grignard bacterium AUH-JLD49s and its application in the conversion of 3 '-demethyls-arctigenin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106399182B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103509741A (en) * | 2013-08-22 | 2014-01-15 | 河北农业大学 | Blautia sp. AUH-JLD56 and application thereof in conversion of arctigenin |
-
2016
- 2016-10-19 CN CN201610909771.1A patent/CN106399182B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103509741A (en) * | 2013-08-22 | 2014-01-15 | 河北农业大学 | Blautia sp. AUH-JLD56 and application thereof in conversion of arctigenin |
Non-Patent Citations (2)
| Title |
|---|
| 微生物发酵转化牛蒡子的研究;徐非一 等;《天然产物研究与开发》;20071231;第19卷;第595-598页 * |
| 肠道微生物对牛蒡苷的转化及转化酶的初步研究;姜洪帅 等;《中国现代中药》;20140131;第16卷(第1期);第9-11页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106399182A (en) | 2017-02-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103509741B (en) | Blautia sp. AUH-JLD56 and application thereof in conversion of arctigenin | |
| CN102367424B (en) | Coriobacterium sp. AUH-Julong21 and use of coriobacterium sp. AUH-Julong21 in liquiritigenin conversion | |
| CN106434783B (en) | A kind of chromone compound and preparation method thereof and the application in preparation antibacterials | |
| CN107298672B (en) | Application of seclenic acid I derived from penicillium oxalicum in preparation of anti-human colon cancer drugs | |
| CN102311981A (en) | Method for preparing and purifying prodigiosin | |
| CN110527629A (en) | A kind of brefeldin A in marine fungi source, preparation method and its application in terms of anti-agriculture pathogenic bacteria | |
| CN101234951B (en) | Biphenyls compound and its preparation method and application | |
| CN101928671B (en) | Alternaria spp and method thereof for preparing ginsenoside Rg3 from fermented ginseng stem-leaf total saponin | |
| CN108315264A (en) | A kind of polyketide in sea paint endogenetic fungus source and its application in preparing anti-inflammatory drug | |
| CN107099464A (en) | A kind of sclerotium aspergillus and its method for preparing penicillic acid | |
| CN103981104A (en) | Endophytic fungi and method thereof for bio-transforming glycyrrhizinic acid into liquiritin | |
| CN103910701A (en) | Marine fungus-derived naphthoquinone compound, and preparation method and application thereof | |
| CN112358516A (en) | Application of diosmetin (4-O-methyl) glucoside compound in preparation of lipid-lowering drugs | |
| CN106399182B (en) | Like grignard bacterium AUH-JLD49s and its application in the conversion of 3 '-demethyls-arctigenin | |
| CN106282032B (en) | Penicillium citrinum LB and the application in phillygenol is prepared in bioconversion forsythin | |
| CN106754408B (en) | One plant of porous trichoderma strain and its method for preparing terpenoid | |
| CN102757443B (en) | Sulfur-substituted podophyllum derivative and bioconversion, separation and purification method thereof | |
| CN107954839A (en) | A kind of antiinflammatory active compound peniroquesine A and its preparation method and application | |
| CN107686492A (en) | A kind of method of rhodioside in extraction purification zymotic fluid using macroporous absorbent resin | |
| CN114213428A (en) | Indole alkaloid compound and preparation method and application thereof | |
| CN102286565A (en) | Preparation method of theaflavin monomer | |
| CN103233056B (en) | Preparation method for great burdock fruit extract through microbial combined fermentation | |
| CN107226801A (en) | A kind of xanthone classes compound and its preparation method of monocrystalline and the application as alpha-glucosidase restrainer | |
| CN102234669B (en) | Biotransformation and purification method of 4-(2,3,5,6-tetramethylpyrazine-1-group)-4'-demethylepipodophyllotoxin | |
| CN110368389A (en) | The preparation method and applications and Claritin of phenolic compound |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |