CN106399182A - Eggerthella sp. AUH-JLD49s and application thereof to conversion of 3'-demethylated-arctigenin - Google Patents

Eggerthella sp. AUH-JLD49s and application thereof to conversion of 3'-demethylated-arctigenin Download PDF

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CN106399182A
CN106399182A CN201610909771.1A CN201610909771A CN106399182A CN 106399182 A CN106399182 A CN 106399182A CN 201610909771 A CN201610909771 A CN 201610909771A CN 106399182 A CN106399182 A CN 106399182A
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aretigenin
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王秀伶
王烨
于飞
刘明月
赵亦楷
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Hebei Agricultural University
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Abstract

The invention discloses Eggerthella sp. AUH-JLD49s and application thereof to the conversion of 3'-demethylated-arctigenin and belongs to the technical field of bacteria. The preservation number of the Eggerthella sp. AUH-JLD49s is CGMCC No. 13124. The application of the Eggerthella sp. AUH-JLD49s includes the steps of firstly, culturing the strain AUH-JLD49s; secondly, co-culturing crude 3'-demethylated-arctigenin serving as the substrate and the strain AUH-JLD49s and performing separation and purification on the converted product. The Eggerthella sp. AUH-JLD49s has the advantages that the strain converts the microbial converted product 3'-demethylated-arctigenin of the arctigenin in traditional Chinese medicine fructus arctii into 3'-demethylated-4'-dehydroxylated-arctigenin, the problem of 3'-demethylated-4'-dehydroxylated-arctigenin resource shortage is solved, and researches and development of 3'-DM-4'-DH-AG pharmacological activity and new drugs are promoted greatly.

Description

Like grignard bacterium AUH-JLD49s and its in 3 '-demethyl-aretigenin conversion Application
Technical field
The present invention relates to bacteria technology field.
Background technology
Fructus Arctii（Arctium lappaL.）For Compositae biennial tuberses medicine food dual purpose plant, its root and stem and leaf can conducts Vegetable eats, and root, fruit and seed also can be used as medicine.Fructus Arctii is the dry mature fruit of Fructus Arctii, has antiinflammatory, anticancer, resists The multiple pharmacological effect such as HIV, anti-diabetic.Wherein Arctiin（Arctiin）It is the main active of Chinese medicine Fructus Arctii（Jilin Normal university's journal, 2011, 11(4): 64-65）.Aretigenin（Arctigenin, abbreviation AG）Slough Fructus Vitis viniferae for Arctiin The free aglycone forms of sugar.Research shows, Arctiin can efficiently produce aretigenin through acid hydrolysis（Contemporary Chinese Chinese medicine, 2012, 14(6): 43-45）.
After Fructus Arctii is taken in vivo by people or other animals, the main active Arctiin in Fructus Arctii will be resided in intestinal Microorganism species be converted into different products.According to the report in 1992 such as Japanese scholars Mitsuhiko Nose, mouse intestinal bacterium Substrate aretigenin can be converted into 3 '-demethyl-aretigenin by group（Planta Medicine, 1992, 58(6):520- 523）；2003, Arctiin was co-cultured by Masao Hattori research group with human faecal mass, the metabolism of 6 kinds of Arctiin is detected Product, product 3 '-demethyl -4 that metabolite 4 therein is in the present invention '-remove hydroxyl-aretigenin.However, due to Masao Hattori etc. uses the microorganism species conversion in human faecal mass, metabolite 4 ability only in a certain special time Can detect（Co-culture 25-125 hour）, the concentration of metabolite 4 is after co-cultivation reaches peak in about 75 hours about Rapid decline again.It is known that compared with single bacterial strain, unstable result when being converted using faecal bacterial community, changing effect Poor reproducibility；In addition, when Masao Hattori etc. utilizes people's faecal bacterial community conversion Arctiin, metabolite species is many, and metabolism is produced Thing 4 grade mesostate is only capable of just detecting in a certain special time period, is difficult to the centre including metabolite 4 Metabolite is effectively prepared（Chemical and Pharmaceutical Bulletin, 2003, 51(4): 378- 384）；2007, Masao Hattori research group separated from people's fresh excrement sample and obtains one plant of Eubacterium bacterial strainEubacteriumsp.ARC-2, substrate aretigenin can be converted into 7 kinds of different nor- based products by strains A RC-2 （Biological and Pharmaceutical Bulletin, 2007, 30(5):904-911）；2013, this laboratory Separate and obtain one plant of Blaw spy's Pseudomonas bacterial strainBlautiaSp. AUH-JLD56, substrate aretigenin can efficiently be turned by this bacterial strain Turn to a kind of product of 3 '-demethyl-aretigenin（Journal Agricultural Food Chemistry, 2013, 61 (49):12060-12065）.The method of strains A UH-JLD56 and its conversion preparation 3 '-demethyl-aretigenin was in 2013 years Shens Report national inventing patent, and be authorized in 2015（ZL 201310368975.5）.The present invention is to authorize patent of invention Product 3 '-demethyl-aretigenin（ZL 201310368975.5）For substrate, separate from people's fresh excrement sample and obtain one plant of energy By specific for 3 '-demethyl-aretigenin be converted into 3 '-demethyl -4 '-remove hydroxyl-aretigenin（3′-DM-4′-DH-AG）'s Bacterial isolateses, and with this bacterial isolates for enzyme source, by microbe transformation method obtain product 3 '-demethyl -4 '-remove hydroxyl Base-aretigenin.
For finding, there is the Structures of Natural Products analog of antiviral activity, German scholar Eich in 1996 etc. is with aryl two Thiophene alkane is starting material, using a series of analog 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% about（Journal of Medicinal Chemistry, 1996, 39, 86-95）.The catalyst Raney's nickel used in above-mentioned chemosynthesis is carcinogenic Thing, triphenylphosphine and butyl lithium are to have irritating danger catalyst, so far, there is no any company both at home and abroad using chemistry Method produces or sells 3 '-DM-4 '-DH-AG.Due to compound 3 '-DM-4 '-DH-AG scarcity of resources, have related compounds 3 '- DM-4 '-DH-AG physiologically active aspect report is few.Only one document report at present, when 3 '-DM-4 '-DH-AG concentration is 10-8 Mol/L（I.e. 10-2Micromoles per liter）When, product 3 '-DM-4 '-DH-AG can remarkably promote MCF-7 cell strainHJ2mm Growth, and work as concentration and be increased to 10-7-10-5During 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）.
Content 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 3 '-demethyl-aretigenin conversion, a kind of conversion of aretigenin in Chinese medicine Fructus Arctii can be produced by this bacterial strain Thing 3 '-demethyl-aretigenin（3′-DMAG）Be converted into 3 '-demethyl -4 '-remove hydroxyl-aretigenin（3′-DM-4′-DH- AG）, solve the problems, such as aretigenin converted product 3 '-DM-4 '-DH-AG resource shortage, 3 '-DM-4 '-DH-AG pharmacology lived Property and new drug development have great impetus.
For solving above-mentioned technical problem, the technical solution used in the present invention is：
A kind of love grignard bacterium AUH-JLD49s（Eggerthellasp.）, preserving number is CGMCC No.13124.
Love grignard bacterium AUH-JLD49s（KX650621）It is that one plant of Gram-negative that separation obtains from human excrement sample is strict Anaerobic bacteria bacterial strain, this bacterial strain can be by 3 '-demethyl-aretigenin in anaerobism work station（3′-DMAG）It is converted into 3 '-nor- Base -4 '-remove hydroxyl-aretigenin（3′-DM-4′-DH-AG）.Relevant 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 2016 October 19 is in China Committee for Culture Collection of Microorganisms's common micro-organisms center（Abbreviation CGMCC）Preservation, preservation address： Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preserving number is CGMCC No.13124.
Love grignard bacteriumEggerthellaThe taxonomy of sp.AUH-JLD49s bacterium is characterized as：
1.0～2.0 millimeters of colony diameter, colony edge is neat, and there is projection at middle part, extends bacterium colony yellow with incubation time and deepens； In BHI culture medium, thalline is shaft-like, and Gram’s staining is negative.
Present invention also offers application in 3 '-demethyl-aretigenin conversion for strains A UH-JLD49s, including following Step：
（1）The culture of strains A UH-JLD49s；
（2）Substrate 3 '-demethyl-aretigenin crude product is co-cultured with strains A UH-JLD49s, by substrate 3 '-demethyl-Fructus Arctii Aglycon crude product be converted into 3 '-demethyl -4 '-remove hydroxyl-aretigenin；
（3）The the isolating and purifying of converted product.
Wherein, the cultural method of strains A UH-JLD49s is：Strains A UH-JLD49s that freezing is preserved is melted simultaneously It is inoculated into by the inoculum concentration of 15-20% volume ratio in the test tube filling fresh BHI fluid medium, 37 DEG C in anaerobism work station Lower culture 18-24 hour；Again strains A UH-JLD49s in test tube is again transferred to by Sheng with the inoculum concentration of 10-15% volume ratio Have in fresh BHI fluid medium, continue to cultivate 18-24 hour in anaerobism work station as seed liquor.
Substrate 3 '-demethyl-aretigenin crude product and strains A UH-JLD49s co-culture method are：
The seed liquor of strains A UH-JLD49s is transferred to culture in BHI fluid medium by the inoculum concentration of 10-15% volume ratio, It is subsequently adding 3 '-demethyl-aretigenin crude product so that 3 '-demethyl-aretigenin concentration in the medium is less than 1.0 mM/ls, cultivate 2-3 days in anaerobism work station, you can by substrate 3 '-demethyl-aretigenin crude product Efficient Conversion For 3 '-demethyl -4 '-remove hydroxyl-aretigenin（3′-DM-4′-DH-AG）.
The mass percent of 3 '-demethyl-aretigenin and 3 '-demethyl-aretigenin crude product is not less than 90%.
In the substrate 3 '-demethyl of direct fermentation-aretigenin crude product, the concentration of 3 '-demethyl-aretigenin is 100- 200 mM/ls.
The the isolating and purifying using following methods of converted product：
With isopyknic ethyl acetate by step（2）In the culture that obtains extract 2 times, extract is evaporated after filtering, and adds 100% methanol solution, with prepare post carry out separating on HPLC prepare 3 '-demethyl -4 '-remove hydroxyl-aretigenin（3′-DM- 4′-DH-AG）.
Wherein, the separation and Culture of strains A UH-JLD49s comprises the steps：
（1）The collection of human excrement sample and culture
With sterilized cotton swab picking fresh excreta, put in 1 milliliter of fresh BHI fluid medium, put in anaerobism work station Cultivate 24 hours at a temperature of 37 DEG C, as the microorganism species of screening microorganisms with specific functions bacterial strain；
（2）Separation and Culture AUH-JLD49s
1. single bacterium colony separation and Culture
With fresh BHI fluid medium, the microorganism species cultivated 24 hours in anaerobism work station in advance are carried out gradient dilute Release, being diluted to concentration respectively is 10–1, 10–2、10–3、10–4、10–5、10–6、10–7、10–8, more respectively by 100 microlitres of concentration respectively For 10–5、10–6、10–7、10–8Microorganism species diluent be evenly coated on the BHI solid medium being made ready beforehand for, will scribble The BHI solid medium of microorganism species diluent is placed in after cultivating 48 hours in anaerobism work station, respectively from BHI solid culture On base, tens of to the hundreds of single bacterium colony of picking is put on BHI solid culture ware, and carries out random number to the single bacterium colony of institute's picking.
2. single bacterium colony activity of conversion measures
Numbered single bacterium colony is inoculated into respectively in the test tube filling 1 milliliter of BHI fluid medium, adds 0.1 mM/l 3 '-demethyl-aretigenin crude product, cultivates 2 days in anaerobism work station, takes 100 microlitres of culture fluid and with 1000 microlitres of acetic acid Ethyl ester is extracted, and extract adds 100% methanol after being evaporated, and is detected with HPLC, finally determines with conversion of substrate The single bacterium colony of 3 '-demethyl-aretigenin function.
The preparation of substrate 3 '-demethyl-aretigenin crude product：
The patent of invention of mandate " the Blaw spy bacterium AUH-JLD56 that the preparation of 3 '-demethyl-aretigenin obtains with this laboratory And its application in aretigenin conversion "（ZL 201310368975.5）.Authorize national inventing patent different from above-mentioned It is that in the present invention, substrate 3 '-demethyl-aretigenin used is to have authorized patent of invention（ZL 201310368975.5）In not Purified 3 '-demethyl-aretigenin crude product（I.e. step（2）In the culture that obtains）.Therefore, patent of invention can be saved （ZL 201310368975.5）In metabolite purification procedures, save time and cost.
The mass percent of 3 '-demethyl-aretigenin and 3 '-demethyl-aretigenin crude product is not less than 90%.
The present invention carries out purification to the product 3 '-DM-4 '-DH-AG obtaining, and first its Anticancer Activity in vitro is ground Study carefully.Result shows, concentration be 50-200 micromoles per liter product 3 '-DM-4 '-DH-AG to human colon cancer cell strain HCT116 and The growth of Breast cancer lines MDA-MB-231 is respectively provided with obvious inhibitory action, wherein concentration be 100 micromoles per liter 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 '- Deepening continuously of DH-AG physiological function research, very likely develops 3 '-DM-4 '-DH-AG for new type anticancer medicine from now on.
Have the beneficial effects that using produced by technique scheme：
Strains A UH-JLD49s that the present invention provides can be by a kind of converted product 3 ' of aretigenin in Chinese medicine Fructus Arctii-nor- Base-aretigenin be converted into 3 '-demethyl -4 '-remove hydroxyl-aretigenin, solve aretigenin converted product 3 '-nor- Base -4 '-go the problem of hydroxyl-aretigenin resource shortage, aretigenin pharmacological active substance microbial metabolic products are ground Study carefully and new drug development has great impetus.
Brief description
The present invention is further detailed explanation with reference to the accompanying drawings and detailed description；
Fig. 1 is the high-efficient liquid phase color of strains A UH-JLD49s conversion of substrate 3 '-demethyl-aretigenin in the embodiment of the present invention 1 Spectrogram；
Fig. 2 is the ultraviolet absorpting spectrum of 3 '-demethyl-aretigenin converted product；
Fig. 3 is the mass spectrum of 3 '-demethyl-aretigenin converted product；
Fig. 4 is the conversion dynamic curve diagram to substrate 3 '-demethyl-aretigenin for the anaerobism bacterial strain AUH-JLD49s；
Fig. 5 is anaerobism bacterial strain AUH-JLD49s to variable concentrations substrate 3 '-demethyl-aretigenin conversion capability comparison diagram；
Fig. 6 be product 3 '-demethyl -4 '-remove hydroxyl-aretigenin to human colon cancer cell strain HCT116 and human breast cancer cell The suppression ratio figure of strain MDA-MB-231.
Specific embodiment
Embodiment 1
1. the separation and Culture of strains A UH-JLD49s
（1）The collection of human excrement sample and culture
With sterilized cotton swab picking people's fresh excreta, put in 1 milliliter of fresh BHI fluid medium, put in anaerobism work station Cultivate 24 hours at a temperature of 37 DEG C, as the microorganism species of screening microorganisms with specific functions bacterial strain.
（2）Separation and Culture strains A UH-JLD49s
1. single bacterium colony separation and Culture
With fresh BHI fluid medium, the microorganism species cultivated 24 hours in anaerobism work station in advance are carried out gradient dilute Release, being diluted to concentration respectively is 10–1, 10–2、10–3、10–4、10–5、10–6、10–7、10–8, more respectively by 100 microlitres of concentration respectively For 10–5、10–6、10–7、10–8Microorganism species diluent be evenly coated on the BHI solid medium being made ready beforehand for, will scribble The BHI solid medium of microorganism species diluent is placed in after cultivating 48 hours in anaerobism work station, respectively from BHI solid culture On base, tens of to the hundreds of single bacterium colony of picking is put on BHI solid culture ware, and carries out random number to the single bacterium colony of institute's picking.
2. single bacterium colony activity of conversion measures
Single bacterium colony numbered, that culture is on BHI solid medium is inoculated into the examination filling 1 milliliter of BHI fluid medium In pipe, then it is separately added into final concentration of 0.1 mM/l of 3 '-demethyl-aretigenin, cultivate 2 days in anaerobism work station, Take 100 microlitres of culture fluid and extracted with 1000 microlitres of ethyl acetate, extract adds 100% methanol after being evaporated, and is examined with HPLC Survey has or not product and generates.Once culture has been detected product generation in certain test tube, just purification culture and bacterium are carried out to bacterial strain Plant preservation, purification and Structural Identification are carried out to product simultaneously.
2. the purification culture of strains A UH-JLD49s, strain identification and preservation
（1）The purification culture of single bacterium colony
The simple function filtering out microbe colony is streak culture on BHI solid medium, after growing single bacterium colony, then to length The single bacterium colony going out is rule, and repeats at least more than 3 times it is ensured that the single bacterium colony form growing is consistent.By single bacterium colony after purification It is inoculated in 1 milliliter of BHI fluid medium, culture takes 100 microlitres of bacterium solution for 24 hours, be added to and fill 500 microlitres and go out in advance In the cryopreservation tube of the defatted milk powder of bacterium, after mixing, add 2 millimeters thick sterilized liquid paraffin in advance on its surface, then by its preservation In -80 DEG C of ultra cold storage freezer, rejuvenation culture is periodically carried out to the functional microorganism bacterial strain of low-temperature preservation and activity of conversion is surveyed Fixed.
（2）Strain identification is carried out to the microorganisms with specific functions bacterial strain isolated
With simple function microbial strains thalline STb gene as template, with universal primer 27F/1492R（27F: 5′- AGAGTTTGATCCTGGCTCAG-3′；1492R: 5′-GGTTACCTTGTTACGACTT-3′）For primer pair 16S rDNA sequence Enter performing PCR amplification, pcr amplification product is delivered Shanghai Sheng Gong biological engineering company limited and carried out DNA sequencing, sequencing result warp BLAST compares and carries out similarity analysis with other bacterial strains of GenBank data base, the 16S rDNA of this simple function microbial strains Sequence and slow love grignard bacteriumEggerthella lentaSimilarity be up to 99.8%, therefore, will be tentatively true for this conversion bacterial strain It is set to love grignard Pseudomonas bacterial strain.This functional microorganism Strain Designation is AUH-JLD49s, and its 16S rDNA sequence is submitted NCBI gene bank, obtains this bacterial strain number of registration（Accession Number）KX650621.
3. application in 3 '-demethyl-aretigenin conversion for strains A UH-JLD49s
（1）The culture of strains A UH-JLD49s
After people's intestinal isolated strains AUH-JLD49s freeze thawing of 80 DEG C of cryopreservation, it is inoculated into by the inoculum concentration of 20% volume ratio Fill in the test tube of fresh BHI fluid medium, 37 DEG C in anaerobism work station at after culture 18 hours in test tube bacterium solution be in all Even muddiness.With the inoculum concentration of 10% volume ratio, muddy strains A UH-JLD49s long in test tube is transferred to again again and fills newly In the teat glass of fresh BHI fluid medium, continue to cultivate 24 hours in anaerobism work station as seed liquor.
（2）Substrate 3 '-demethyl-aretigenin crude product is co-cultured with strains A UH-JLD49s
The seed liquor of above-mentioned cultured in advance strains A UH-JLD49s is pressed the inoculum concentration of 10% volume ratio in anaerobism work station It is transferred to the triangle culture in glassware filling fluid medium, the concentration being simultaneously introduced 3 '-demethyl-aretigenin is 150 mmoles You/liter 3 '-demethyl-aretigenin crude product so that 3 ' in culture medium-demethyl-aretigenin concentration be 1.0 mMs/ Rise, cultivate 3 days in anaerobism work station, substrate 3 '-DMAG can be converted into 3 '-DM-4 '-DH-AG by strains A UH-JLD49s.
（3）With HPLC detection substrate 3 '-demethyl-aretigenin by the conversion situation of strains A UH-JLD49s
Take out in 100 microlitres to 1.5 milliliters EP pipes of culture from above-mentioned triangular flask, plus 1000 microlitres of ethyl acetate are extracted Take, after standing centrifugation, take out 800 microlitres of upper strata ethyl acetate, after being evaporated in centrifuge concentrator, add 800 microlitre of 100% methanol Solution, converts situation through HPLC detection substrate（See accompanying drawing 1）.
（4）The the isolating and purifying of converted product
If good through HPLC detection substrate conversion, with isopyknic ethyl acetate, culture in triangular flask is extracted 2 times, extraction Liquid is evaporated after filtering on a rotary evaporator, is subsequently adding 100% methanol solution, uses after crossing the organic membrane that aperture is 0.45 micron Prepare post to carry out separately preparing on HPLC, collect metabolite 3 '-DM-4 '-DH-AG with clean triangular flask.
Embodiment 2
, with embodiment 1, strains A UH-JLD49s is in 3 '-demethyl-aretigenin for the isolated culture method of strains A UH-JLD49s Application in conversion comprises the steps：
（1）The culture of strains A UH-JLD49s
After people's intestinal isolated strains AUH-JLD49s freeze thawing of 80 DEG C of cryopreservation, it is inoculated into by the inoculum concentration of 15% volume ratio Fill in the test tube of fresh BHI fluid medium, 37 DEG C in anaerobism work station at after culture 20 hours in test tube bacterium solution be in all Even muddiness.With the inoculum concentration of 15% volume ratio, muddy strains A UH-JLD49s long in test tube is transferred to again again and fills newly In the teat glass of fresh BHI fluid medium, continue to cultivate 18 hours in anaerobism work station as seed liquor.
（2）Substrate 3 '-demethyl-aretigenin crude product is co-cultured with strains A UH-JLD49s
The seed liquor of above-mentioned cultured in advance strains A UH-JLD49s is pressed the inoculum concentration of 15% volume ratio in anaerobism work station It is transferred to the triangle culture in glassware filling fluid medium, the concentration being simultaneously introduced 3 '-demethyl-aretigenin is 100 mmoles You/liter 3 '-demethyl-aretigenin crude product so that in culture medium concentration of substrate be 0.8 mM/l, in anaerobism work station Interior culture 2.5 days, substrate 3 '-DMAG can be converted into 3 '-DM-4 '-DH-AG by strains A UH-JLD49s.
（3）HPLC detection substrate 3 '-demethyl-aretigenin is by the conversion situation of strains A UH-JLD49s
Take out in 100 microlitres to 1.5 milliliters EP pipes of culture from above-mentioned triangular flask, plus 1000 microlitres of ethyl acetate are extracted Take, after standing centrifugation, take out 800 microlitres of upper strata ethyl acetate, after being evaporated in centrifuge concentrator, add 800 microlitre of 100% methanol Solution, converts situation through HPLC detection substrate.
（4）The the isolating and purifying of converted product
If good through HPLC detection substrate conversion, with isopyknic ethyl acetate, culture in triangular flask is extracted 2 times, extraction Liquid is evaporated after filtering on a rotary evaporator, is subsequently adding 100% methanol solution, uses after crossing the organic membrane that aperture is 0.45 micron Prepare post to carry out separately preparing on HPLC, collect metabolite 3 '-DM-4 '-DH-AG with clean triangular flask.
Embodiment 3
, with embodiment 1, strains A UH-JLD49s is in 3 '-demethyl-aretigenin for the isolated culture method of strains A UH-JLD49s Application in conversion comprises the steps：
（1）The culture of strains A UH-JLD49s
After people's intestinal isolated strains AUH-JLD49s freeze thawing of 80 DEG C of cryopreservation, it is inoculated into by the inoculum concentration of 18% volume ratio Fill in the test tube of fresh BHI fluid medium, 37 DEG C in anaerobism work station at after culture 24 hours in test tube bacterium solution be in all Even muddiness.With the inoculum concentration of 13% volume ratio, muddy strains A UH-JLD49s long in test tube is transferred to again again and fills newly In the teat glass of fresh BHI fluid medium, continue to cultivate 20 hours in anaerobism work station as seed liquor.
（2）Substrate 3 '-demethyl-aretigenin crude product is co-cultured with strains A UH-JLD49s
The seed liquor of above-mentioned cultured in advance strains A UH-JLD49s is pressed the inoculum concentration of 12% volume ratio in anaerobism work station It is transferred to the triangle culture in glassware filling fluid medium, the concentration being simultaneously introduced 3 '-demethyl-aretigenin is 200 mmoles You/liter 3 '-demethyl-aretigenin crude product so that in culture medium concentration of substrate be 0.6 mM/l, in anaerobism work station Interior culture 2 days, substrate 3 '-DMAG can be converted into 3 '-DM-4 '-DH-AG by strains A UH-JLD49s.
（3）With HPLC detection substrate 3 '-demethyl-aretigenin by the conversion situation of strains A UH-JLD49s
Take out in 100 microlitres to 1.5 milliliters EP pipes of culture from above-mentioned triangular flask, plus 1000 microlitres of ethyl acetate are extracted Take, after standing centrifugation, take out 800 microlitres of upper strata ethyl acetate, after being evaporated in centrifuge concentrator, add 800 microlitre of 100% methanol Solution, converts situation through HPLC detection substrate.
（4）The the isolating and purifying of converted product
If good through HPLC detection substrate conversion, with isopyknic ethyl acetate, culture in triangular flask is extracted 2 times, extraction Liquid is evaporated after filtering on a rotary evaporator, is subsequently adding 100% methanol solution, uses after crossing the organic membrane that aperture is 0.45 micron Prepare post to carry out separately preparing on HPLC, collect 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, authentication method and qualification result are as follows：
After the converted product peak of collection is evaporated, its purity is measured on analytical type high performance liquid chromatograph, then measure generation respectively Thank ultraviolet absorption spectrum, mass spectrum, proton nmr spectra and the carbon spectrum of product.Result shows, this converted product respectively at 228 nanometers and 278 nanometers have maximal ultraviolet absorption（See accompanying drawing 2）, mass spectroscopy result shows, this metabolite molecular weight is 342（See attached Fig. 3）, this lucky and 3 '-DM-4 '-DH-AG molecular formula C20H22O5Match.Ultraviolet absorpting spectrum according to product and molecular weight 3 '-DM-4 '-DH-AG can be initially identified as.For accurately parsing the chemical constitution of this metabolite further, we are respectively Determine the proton nmr spectra of metabolite after purification（1H-NMR）With carbon spectrum（13C-NMR）.By spectrum elucidation, and with literary composition Offer the proton nmr spectra of 3 '-DM-4 '-DH-AG in report and carbon spectrum is analyzed（Chemical andPharmaceutical Bulletin,2003, 51(4):378-384）, the most at last this metabolite precise Identification be 3 '- DM-4′-DH-AG.Metabolite1H-NMR and13C-NMR result is as follows：
1H NMR (CDCl3, 400 MHz):δ2.41-2.55 (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):δ179.1 (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′).
Analysis strains A UH-JLD49s converts dynamic and conversion capability to substrate 3 '-demethyl-aretigenin crude product, and Product 3 '-demethyl -4 '-remove hydroxyl-aretigenin to human colon cancer cell strain HCT116 and Breast cancer lines MDA-MB- 231 inhibitory action：
1st, strains A UH-JLD49s is dynamic to the conversion of substrate 3 '-demethyl-aretigenin
For understanding the speed of strains A UH-JLD49s conversion of substrate 3 '-demethyl-aretigenin, so that when determining optimal conversion Between, we are dynamically determined to the conversion of strains A UH-JLD49s conversion of substrate 3 '-demethyl-aretigenin, specifically side Method is as follows：
To cultured seed liquor in advance（Cultural method is same as above）It is inoculated into by the inoculum concentration of 10% volume ratio and fill 100 millis Rise 250 milliliters of triangle culture in glassware of fluid medium, the concentration being simultaneously introduced 3 '-demethyl-aretigenin is 150 mmoles You/liter 3 '-demethyl -0.4 milliliter of aretigenin crude product, in anaerobism work station culture 0 hour, 3 hours, 6 hours, 9 little When, 12 hours, 15 hours, 18 hours, 21 hours, take 100 microlitres of culture after 24 hours respectively, with 1000 microlitres of acetic acid second Ester extracts, and takes 800 microlitres of extract, and extract adds 100 microlitre of 100% methanol solution after being evaporated, and with HPLC detection substrate quilt Conversion situation, draws bacterial strain conversion according to different incubation time substrates and production concentration dynamic（See Fig. 4）, as shown in Figure 4, substrate 84.7% about product 3 '-DM-4 '-DH-AG is had to be generated after adding 12 hours, after continuing culture 15 hours, the bottom that added Thing aretigenin has 96.8% to be converted into 3 '-DM-4 '-DH-AG, and product amount no longer rises substantially within the 24th hour.
2nd, strains A UH-JLD49s measures to variable concentrations substrate 3 '-demethyl-aretigenin conversion capability
By strains A UH-JLD49s respectively with the 3 '-demethyl-aretigenin of variable concentrations in anaerobism work station co-cultivation 3 My god, then with equal-volume ethyl acetate, culture is extracted, extract adds 100% methanol solution after being evaporated, and uses HPLC Detection substrate 3 '-demethyl-aretigenin crude product is converted situation.Result shows, when substrate 3 '-demethyl-aretigenin is dense When degree is less than 1.0 mM/ls, strains A UH-JLD49s is average to the substrate 3 '-demethyl-aretigenin of variable concentrations Conversion ratio（Conversion ratio=production concentration/（Remaining concentration of substrate+production concentration）×100%）It is more than 90%, such as strains A UH- JLD49s is respectively the average of 0.4 mM/l, 0.6 mM/l, 0.8 mM/l and 1.0 mM/ls to concentration Conversion ratio is respectively 97.4%, 93.3%, 90.3% and 90.0%；When concentration of substrate increases to 1.2 mM/ls, strains A UH- JLD49s decreases to the transformation efficiency of substrate mM/l, and average conversion is 84.7%；And it is dense to work as added substrate When spending for 1.6 mM/ls, strains A UH-JLD49s drastically declines to substrate mM/l conversion capability, and average conversion is only For 18.0%（See accompanying drawing 5）.
3rd, product 3 '-demethyl -4 '-go hydroxyl-aretigenin thin to human colon cancer cell strain HCT116 and human breast carcinoma The suppression ratio of born of the same parents strain MDA-MB-231
Take the logarithm each tumor cell of trophophase, with 0.25% trypsinization, then made with RPMI-1640 culture medium Single cell suspension, adjustment cell concentration is 5 × 104Individual/milliliter, by cell suspension inoculation in 96 well culture plates, every hole 100 is micro- Rise, after cell attachment each group be separately added into 50 micromoles per liter, 100 micromoles per liter, 200 micromoles per liter 3 '-demethyl- 4 '-go hydroxyl-aretigenin and blank culture fluid and positive control agent, respectively set 3 multiple holes.Culture plate is placed in CO2Incubator In, cultivate 24 hours each culture hole addition 20 microlitres/holes of MTT backward, after being incubated 4 hours for 37 DEG C（Suspension cell need to be through centrifugation）, Careful suction abandons supernatant culture fluid, and every hole adds 100 microlitres of DMSO, and agitator vibrates 10 minutes, fully dissolving first a ceremonial jade-ladle, used in libation （Formazan）Crystallization, microplate reader detects each hole absorbance A value, measures wavelength and is 570 nanometers, calculates suppression ratio IR=（1- medication Group mean OD value/matched group mean OD value）×100%.Result shows, concentration is the product 3 '-DM- of 50-200 micromoles per liter 4 '-DH-AG are respectively provided with obvious suppression to the growth of human colon cancer cell strain HCT116 and Breast cancer lines MDA-MB-231 Make and use, 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%（See accompanying drawing 6）.

Claims (7)

1. a kind of love grignard bacterium（Eggerthellasp.）AUH-JLD49s, preserving number is CGMCC No.13124.
2. application in 3 '-demethyl-aretigenin conversion for the love grignard bacterium AUH-JLD49s as claimed in claim 1, its It is characterised by comprising the steps：
（1）The culture of strains A UH-JLD49s；
（2）Substrate 3 '-demethyl-aretigenin crude product is co-cultured with strains A UH-JLD49s, by substrate 3 '-demethyl-Fructus Arctii Aglycon crude product be converted into 3 '-demethyl -4 '-remove hydroxyl-aretigenin；
（3）The the isolating and purifying of converted product.
3. application in 3 '-demethyl-aretigenin conversion for the love grignard bacterium AUH-JLD49s according to claim 2, It is characterized in that：Step（1）, the cultural method of strains A UH-JLD49s is：Strains A UH-JLD49s that freezing is preserved Melt and be inoculated into by the inoculum concentration of 15-20% volume ratio in the test tube filling fresh BHI fluid medium, in anaerobism work station 18-24 hour is cultivated at interior 37 DEG C；With the inoculum concentration of 10-15% volume ratio, strains A UH-JLD49s in test tube is turned again again It is connected to and fills in fresh BHI fluid medium, continue to cultivate 18-24 hour in anaerobism work station as seed liquor.
4. application in 3 '-demethyl-aretigenin conversion for the love grignard bacterium AUH-JLD49s according to claim 3, It is characterized in that：Step（2）, substrate 3 '-demethyl-aretigenin crude product and strains A UH-JLD49s co-culture method are：Will The seed liquor of strains A UH-JLD49s is transferred to culture, Ran Houjia in BHI fluid medium by the inoculum concentration of 10-15% volume ratio Enter 3 '-demethyl-aretigenin crude product so that 3 '-demethyl-aretigenin concentration in the medium is less than 1.0 mmoles You/liter, in anaerobism work station cultivate 2～3 days, by substrate 3 '-demethyl-aretigenin crude product Efficient Conversion be 3 '-nor- Base -4 '-remove hydroxyl-aretigenin.
5. the answering in 3 '-demethyl-aretigenin conversion of the love grignard bacterium AUH-JLD49s according to claim 2 or 4 With it is characterised in that：3 '-demethyl-aretigenin is not less than with the mass percent of 3 '-demethyl-aretigenin crude product 90%.
6. application in 3 '-demethyl-aretigenin conversion for the love grignard bacterium AUH-JLD49s according to claim 4, It is characterized in that：In the substrate 3 '-demethyl of direct fermentation-aretigenin crude product, the concentration of 3 '-demethyl-aretigenin is 100-200 mM/l.
7. application in 3 '-demethyl-aretigenin conversion for the love grignard bacterium AUH-JLD49s according to claim 2, It is characterized in that：Step（3）, the isolation and purification method of converted product is：With isopyknic ethyl acetate by step（2）In obtain Culture extract 2 times, extract filter after be evaporated, add 100% methanol solution, with prepare post carry out on HPLC separate make Standby 3 '-demethyl -4 '-remove hydroxyl-aretigenin.
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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

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