CN106967622A - One plant of paclitaxel produced aspergillus flavus BP6T2 and its application - Google Patents
One plant of paclitaxel produced aspergillus flavus BP6T2 and its application Download PDFInfo
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Abstract
The invention discloses one plant of paclitaxel produced endophyte separated from the tissue of taxaceae Bashan Mountain Chinese torreya, aspergillus flavus BP6T2 is named as by taxonomic identification, deposit number is CCTCC NO:M2016318.There is the bacterial strain extractive content in the activity of anti-Staphylococcus aureus and Escherichia coli, zymotic fluid to be 8.4% up to 93mg/L, the wherein percentage contents of taxol, and yield is 7.81mg/L, higher than the published yield of prior art；Aspergillus flavus BP6T2 inhibitory action of the fermentation broth extract to Hela tumour cells is demonstrated using the reagent methods of CCK 8, inhibiting rate can reach 66.20%.
Description
Technical field
The invention belongs to microbial technology field, and in particular to the aspergillus flavus BP6T2 isolated in a kind of Chinese torreya from the Bashan Mountain,
The bacterial strain has the characteristic of High Yield of Taxol, further relates to applications of the aspergillus flavus BP6T2 in treatment tumour cell.
Background technology
Taxol (Paclitaxel) alias PTX, Paclitaxe, Paclitaxe, molecular weight 853.92, molecular formula C47H51NO14(figure
1) it is, cell mitogen inhibitor, is used for treatment of cancer (Wani MC, Taylor HL, Wall ME, et al.Plant
antitumor agents.VI.The isolation and structure of taxol,a novel antileukemic
and antitumor agent from Taxus brevifolia[J].J Am Chem Soc,1971,93:2325~
2327.)。
Taxol and the like is considered as the efficient, cancer therapy drug of wide spectrum, hypotoxicity, and ovary is especially treated at present
Cancer, uterine cancer and the best medicine of breast cancer, also have to lung cancer, colorectal cancer, melanoma, incidence cancer, lymthoma, brain tumor
Certain curative effect.It is a kind of high efficiency cell toxin, compared with the other chemical synthesis antitumor agents or phytogenic anticarcinogen that have listed,
With unique anticancer mechanism.It specifically binds to the vascular bundle albumen of the cell such as cancer cell in division, especially β-micro-
On tubulin, promote tubulin polymerization, suppress depolymerization, keep tubulin stable, tumour cell is stopped at G2 phases and M
Phase, suppress cell mitogen, so as to prevent the propagation of cancer cell, thus gross tumor volume is gradually reduced, rather than directly kill
Dead cancer cell.In tumour cell, taxol, which can induce, promotees apoptogene, such as Bax, p53 and p21, C-Mos, the table of TNF-α etc.
Reach and inducing apoptosis.It has also been found that taxol can strengthen the phosphorylation of EGFR-TK, the signal transduction pathway of active cell apoptosis,
Promote Apoptosis.Taxol can also adjust the immunologic function of body, promote IL-1, the secretion of IFN-α and IFN-β, INF- α etc.
And release, tumour cell is play a part of to kill or suppressed.
Taxol was most found earlier than 1967 in National Cancer Institute, last century the fifties end, state of the U.S.
Family's Cancer Institute has formulated a huge plan that anti-cancer active matter is screened from the plant of the whole America.By the programme support,
Monroe E.Wall and Mansukh C.Wani were carried in 1963 from Pacific Ocean Chinese yew (Taxus brevifolia) bark
Take out taxol crude extract.1966, taxol was purified, and its structure is determined.Then prove that it has higher
Anti- B16 black tumor activity.1979, Schiff and Horwitz illustrated taxol and specifically bind to cell in division such as
Cancer cell.To on December 29th, 1992, FDA (Food and Drug Adminstration) (FDA) just ratified Bristol-Myers-Squibb
(BMS) company lists taxol, trade name Taxol, is mainly used in treatment oophoroma and breast cancer.
But content of taxol is extremely low in the plant such as Taxus, only 0.003%-0.069%.Treat 1 cancer
The amount of taxol required for patient is the bark side for needing to cut down the Chinese yew of 3 plants of growths 60 years, or 1 plant of Chinese yew in thousand
It can obtain.1kg taxols are often extracted, about 2000 a century is cut down and set greatly.And its natural resources Chinese yew and East Africa
Podocarpus grows very slow, limited amount, it is difficult to the need for meeting the mankind, therefore its price is very expensive.Even if felling
Whole natural resources, can only also meet short-term needs, and cause to forest and environment for human survival and bio-diversity not
The loss that can be made up.Therefore, the guarantee of Japanese yew raw polyol just turns into the key factor that the medicine success moves towards market.
In order to solve, medicine source that the factors such as some medicinal plants are slow-growing, resource is nervous bring is nervous and ecological disruption is asked
Topic, people are also attempting to utilize other method production taxols, such as chemical synthesis, semi-chemical synthesis, cell always
Tissue cultures etc., its main feature is as follows：
(1) chemical fully synthetic taxol method obtains success, but whole process is very expensive, and yield is not high, and
A non-desirable route.
(2) although semi-synthetic effective, semi-synthetic precursor is isolated and purified from plant tissue also more difficult.
(3) the step of Plant Tissue Breeding needs is relatively simple, but Taxol Yield is relatively low, and yield is unstable.
The separation of paclitaxel produced or the like endogenetic fungus is that a kind of taxol that effectively solves developed in recent years is provided
The new way of source problem.Taxol is largely produced by the method for microbial fermentation, be expected to improve current taxol it is expensive,
The present situation that supply falls short of demand.Even if its yield is suitable or lower slightly with content of taxol in plant, but because the fungi fermentation cycle is short by (10
~15 days), fermentation-scale can be with manual control, and its production capacity is necessarily significantly better than the production capacity of vegetable material.Therefore, seek
The plant endogenesis epiphyte of taxol can be produced by looking for, and taxol is largely produced by the method for microbial fermentation, be expected to improve mesh
The present situation that preceding taxol is expensive, supply falls short of demand.
Endophyte of plant is widely present in almost various plants, in long-term evolutionary process, they and plant host
Between form harmony ecological relationship.Host provides metastable environment for them, and endophyte then assign host's antibacterial,
The characteristic such as antiviral, drought-resistant and pest-resistant.By long-term common evolutionary, may also can in the endophyte of some medicinal plants
It is enough to produce and the same or analogous active medicinal matter of host.Therefore the endogenetic fungus research of many medicinal plants is received extensively
Concern, including the Separation Research of paclitaxel produced or the like plant endogenesis epiphyte has been affected by extensive concern.
1993, Stierle etc. isolated one kind from mountain mahogany (Taxus brevifolia) bast first
New endophytic fungi --- Andrew's Japanese yew bacterium (Taxomyces andreanae), can produce purple in semi-synthetic nutrient solution
China fir alcohol and bearing taxanes, show that the endogenetic fungus having has synthesis and the same or analogous active component of host plant
Ability.Hereafter, scholars carry out the mask work of taxol-producing endophytic fungi one after another.Li etc. is from bald cypress (Taxodium
Distichum paclitaxel produced endogenetic fungus (Pestalotiopsis is all separated under bark) in bast and xylem
microspora).Strobel etc. is separated to the interior life of one plant of generation taxol from Xizang Taxus chinensis (T.wallachiana)
Fungi Pestalotiopsis microspora (Pestalotiopsis microspora), the nutrient solution of this fungi can produce taxol
Amount reach 50 μ g/L；Hereafter Nepal's disk end deer horn bacterium (Seimatoantlerium nepalense) is separated to again, its is true
The amount that bacteria culture fluid produces taxol is 62~80ng/L.This illustrates that paclitaxel produced endogenetic fungus is not only present in Taxus
In plant, there is also further expand the screening scope of taxol-producing endophytic fungi in some non-Chinese yew genus plants.
Chinese scholar also achieves certain achievement in the research of separation taxol-producing endophytic fungi., Qiu De in 1994
Have etc. and a kind of endogenetic fungus YFI is isolated from taxusyunnanensis (T.yunnanensis) bark, form is similar to Stierle
It was found that Andrew Japanese yew bacterium, analyze and determine by TLC, show also produce taxol, but biosynthesis amount is very low.Zhou Dong
Slope etc. finds a kind of endogenetic fungus from taxus chinensis in northeast (T.cuspidase) --- treelike menu (Nodulisporium
Sp.), taxol can be produced, is the genus new to China of China, yield is 51.06~125.7 μ g/L.Wang Weis etc. are from southern red bean
The isolated 9 plants of endophytes of trunk and side shoot bark and subcutaneous part of China fir (T.chinensisvar mairei), wherein 6 plants
Bearing taxanes can be secreted, identified cephalosporium (Cephalosporium sp.), the wheel shank of being belonging respectively to combs mould category
(Martensiomyces sp.) and without spore bacterium (Mycelia sterlia).The bright grade of Valsartan is also divided from Chinese southerm yew
From to one plant of taxol-producing endophytic fungi, being identified as Fusarium (Fusarium mairei), the μ g/L of yield about 20.
Because paclitaxel produced or the like the content of wild-type fungal is generally relatively low, in order to improve its yield, current grinds
Study carefully and be concentrated mainly on the following aspects：Such as using routine mutagenesis improvement strain, (Zhou Dongpo puts down auspicious, the taxols such as Sun Jianqiu of text
Research [J] JOURNAL OF MICROBIOLOGYs of producing strains separation, 2001,21 (1)：18~21.)；Strain is improved using Protoplast Mutation
(Zhao Kai puts down auspicious, the horse imperial or royal seal etc. of text, the Protoplasts Mutagenesis of taxol superior strain and its hereditary variation pre-test, microbiology
Report, 2005,45 (3), 355-358.)；Engineered strain improvement strain is built using technique for gene engineering；Optimal Medium and fermentation
Condition etc.；These paclitaxel produced or the like fungies are applied in production, how its key issue is its yield.But
The yield for the producing strains having now been found that is not up to the requirement of industrialized production.
So far, taxol-producing endophytic fungi bacterial strain is main separates from the plant of endangered species Taxus, but neglects
Other platymisciums in taxaceae are omited.There are some researches show, it has also been found that there is bearing taxanes in taxaceae Chinese torreya platymiscium,
But content is extremely low, content of taxol is less than 0.003%, it is believed that medicinal Development volue less (grind by Yi Guanmei, Qiu Yingchun Chinese torreyas
Study carefully current situation and development [J] resource and environments, 2013,29 (8)：844-848).Liu Yan etc. is from Cephalotaxus mannii (Cephalotaxus
Hainanensis Li) in be separated to production secondary metabolite strain, the antineoplastic material tricuspid of possibility mainly produced
(the Cephalotaxus mannii endogenetic fungus such as Liu Yan CH1307 is identified China fir ester alkaloid compound and its antitumor activity [D] Hainan is big
Learn, clean Cephalotaxus manniis endogenetic fungus antitumor activity bacterial strain screening [D] the University Of Hainan of 2012. Jiang Chun, 2014.)
From Shennongjia National Geology Forest Park Bashan Mountain Chinese torreya, (taxaceae, Chinese torreya belongs to the present invention；Torreya
Fargesii endogenetic fungus) is separated in tissue, by bacteriostatic test, the endogenetic fungus with antibacterial activity plant is filtered out；Again
Analyzed by efficient liquid phase chromatographic analysis and antitumor activity, filter out the bacterium for producing taxol or the like secondary metabolite
Kind；And by using LC-MS instrument qualitative detection, thus it is speculated that the bearing taxanes that the bacterial strain is produced are taxol.
Taxol is largely produced by the method for microbial fermentation, be expected to improve current taxol it is expensive, for should
The present situation asked.Even if its yield is suitable or lower slightly with content of taxol in plant, but because the fungi fermentation cycle is short by (10~15
My god), fermentation-scale can be with manual control, and its production capacity is necessarily significantly better than the production capacity of vegetable material.
The content of the invention
The purpose of the present invention is the endophyte aspergillus flavus isolated in being the provision of a kind of Chinese torreya from the Bashan Mountain
(Aspergillus flavus) BP6T2, the bacterial strain has anti-Staphylococcus aureus (G+) and Escherichia coli (G-) activity, together
When also have High Yield of Taxol characteristic.
Another object of the present invention is to be the provision of aspergillus flavus (Aspergillus flavus) BP6T2 to prepare treatment
Application in tumour cell medicine, tests prove that, aspergillus flavus BP6T2 fermentation broth extracts have obvious to Hela tumour cells
Inhibitory action.
To achieve the above object, the present invention uses following technical proposals：
Aspergillus flavus (Aspergillus flavus) BP6T2 separation screening, its step is：
1st, the root of Bashan Mountain Chinese torreya, stem, leaf, bark are blotted respectively with after aseptic water washing, successively with 75% (v/v's)
Alcohol disinfecting 5min and 2% (v/v) hypochlorous acid sterilization 8min, then with aseptic water washing, with sterile knife by the root collected, stem
Segment is cut into bark, takes the material at each position to be placed in PDA solid medium plates, is positioned in 28 DEG C of insulating boxs and cultivates, treat
Inoculum tangent plane edge grows and purified on tiny mycelia, the PDA solid plates that picking mycelia is transferred new in above-mentioned plate, until
For pure culture；
2nd, the fungi of purifying is seeded on PDA plate, 28 DEG C of cultures, the fine and close journey of record bacterium colony size, color, mycelia
The colonial morphologies such as whether degree, colony edge are neat, colony growth rate and chromogenic element；
3rd, the bacteriostatic activity of endogenetic fungus is determined with agar block method：Bacterium (staphylococcus aureus, large intestine bar will be tested
Bacterium) with after the activation of LB culture mediums, it is respectively coated on inoculation in LB solid culture primary surfaces, each plate and carries agar medium
Endophyte, 37 DEG C are cultivated 1-2 days, the inhibition zone around observation agar block, and obtaining one plant has while having anti-golden yellow grape
Coccus (G+) and Escherichia coli (G-) activity bacterial strain, be designated as BP6T2；
4th, observed by plate cultural character, the bacterial strain colony colour is in grass green, bacterium colony quality is velvet shape, microscope
Lower its conidial head of observation is in loose radial, and top capsule is subsphaeroidal, and individual layer and double-deck stigma exist simultaneously, are accredited as aspergillus flavus
(Aspergillus flavus)BP6T2；
5th, by strain BP 6T2 in PDA liquid medium, 180rpm, 28 DEG C activate 3 days, as seed liquor, by seed liquor
With 5% (v/v) inoculum concentration, access in PDA liquid medium, 180rpm, 7-10 days harvest zymotic fluids of 28 DEG C of cultures；
6th, after fermentation ends, the ethyl acetate that 1/3 volume is added in filtrate, filtrate, counter-current extraction 3 times, in collection are collected
Layer organic phase, removes organic solvent in Rotary Evaporators, obtains fat-soluble extract, and its content is averagely about 93mg/L；
7th, high performance liquid chromatography (HPLC) is found in extract containing basically identical with Taxol Standard retention time
Composition, measures retention time for 13.208min, close with standard items retention time, shows that it contains taxol or the like；
8th, LC-MS instrument (HPLC-MS) determines the structure of paclitaxel analogs in metabolism extract, shows its parent ion
Da is 876.300, and daughter ion Da is respectively 307.900,591.200 and 531.200, and source voltage is 150, and impact energy is respectively
36th, 36 and 40, it is completely the same with bearing taxanes Taxol Standard, therefore judge aspergillus flavus (Aspergillus
Flavus) the metabolism extract that BP6T2 is produced is taxol.
The endophyte of plant aspergillus flavus (Aspergillus flavus) separated in described Bashan Mountain Chinese torreya tissue
BP6T2, is preserved in China typical culture collection center (CCTCC), preservation address：Chinese Wuhan Wuhan Universitys, preservation day
Phase：On June 8th, 2016, deposit number：CCTCC NO.M2016318, it is characterized in that with anti-Staphylococcus aureus and large intestine
The content of taxol reaches 7.81mg/L in the activity of bacillus, zymotic fluid.
Applications of the aspergillus flavus BP6T2 in treatment tumour cell medicine is prepared：Aspergillus flavus BP6T2 is determined by CCK-8 methods
Fermentation broth extract to the inhibitory action of Hela cells, as a result show the rise of the concentration with extract, cell proliferation
Inhibitory action enhancing, wherein with 3.104 μ g/mL concentration handle inhibitory action it is most strong, inhibiting rate is 66.20%, thus speculate
The anti-Hela cellular materials that aspergillus flavus BP6T2 is produced are taxol.
Compared with prior art, the present invention has advantages below and beneficial effect：
1st, so far, taxol-producing endophytic fungi bacterial strain is main plants from the plant of endangered species Taxus and podocarpus
Separated in thing, studied the endophyte also isolated from bald cypress and produce taxol.The present invention is also from beyond Taxus
Plant in as isolated paclitaxel produced endogenetic fungus in Bashan Mountain Chinese torreya (taxaceae, Chinese torreya category) tissue, further expand
The scope of resource for producing taxol or the like strain is obtained, is conducive to broadly utilizing microbial resources.
2nd, this research is to obtain the aspergillus flavus for producing paclitaxel analog compound first, the endophyte of plant aspergillus flavus BP6T2
Paclitaxel analogs can be very stably produced, fermentation broth extract can reach 93mg/L, and taxol percentage contains
Measure as 10.5%, the yield of taxol is 7.81mg/L, higher than a kind of published yield of prior art (Yunnan of the such as Qiu Deyou
The separation of Chinese yew endogenetic fungus,《Fungus journal》,1994(4):314-316；Li J,et al.Ambuic acid,a
highly functionalized cyclohexenone with antifungal activity Pestalotiopsis
spp.and Monochaetia sp Phytochemistry,2001,56(5):463-468；Zhou Dongpo, puts down auspicious, the Sun Jianqiu of text
Research [J] the JOURNAL OF MICROBIOLOGYs separated Deng Taxol-producing fungis, 2001,2l (1)：18—20；Zhao Kai, puts down auspicious, the Ma Xi of text
Deng the Protoplasts Mutagenesis of taxol superior strain and its hereditary variation pre-test, microorganism journal, 2005,45 (3)：
355-358.)。
3rd, suppression of the metabolism extract to Hela tumour cells in aspergillus flavus BP6T2 zymotic fluids is examined using CCK-8 reagents method
Make and use, experiment shows the rise with extract concentrations, the inhibitory action to Hela tumor cell proliferations strengthens, when its concentration
Inhibiting rate is 39.90% during for 0.0310 μ g/mL, when with 3.104 μ g/mL concentration handle Hela cells inhibitory action it is most strong,
Inhibiting rate is 66.20%.
4th, can not be by resource, environment, condition, equipment etc. using the endophyte of plant fermenting and producing taxol of the present invention
Various limitations, the short time, produced on a large scale.
Brief description of the drawings
Fig. 1 is the chemical structural formula of taxol.
Fig. 2 is aspergillus flavus BP6T2 lawn photo, and colony colour is in grass green, and bacterium colony quality is velvet.
Fig. 3 is aspergillus flavus BP6T2 microphoto, and conidial head is in loose radial, and top capsule is subsphaeroidal, stigma individual layer
It is double-deck to exist simultaneously.
Fig. 4 is the high-efficient liquid phase chromatogram of Taxol Standard, and peak shown in arrow is taxol.
Fig. 5 is the high-efficient liquid phase chromatogram that aspergillus flavus BP6T2 is metabolized extract, and peak shown in arrow is paclitaxel analogs.
Embodiment
Embodiment 1：Bashan Mountain Chinese torreya endophyte of plant is isolated and purified
1st, the pretreatment of Bashan Mountain Chinese torreya material
Root, stem, leaf, the bark of Bashan Mountain Chinese torreya are gathered in Hubei Province Shennongjia National Geology Forest Park, is rushed with sterilized water
After washing, blotting paper is blotted；Alcohol disinfecting 5min and the sterilization of 2% (v/v) hypochlorous acid 8min, Ran Houyong respectively through 75% (v/v)
The root collected, stem and bark are cut into about 2-3cm segments by aseptic water washing with sterile knife again, and blade cuts out section from centre.
2nd, the separation of Bashan Mountain Chinese torreya endophyte of plant
The material at each position of pretreatment is taken to be placed in PDA solid medium plates, in 28 DEG C of cultures；PDA culture medium：Ma Ling
Potato 200g, glucose 20g, KH2PO41.0g, MgSO4·7H2O 0.5g, water 1000mL, plus agar (15g/L) are PDA solids
Culture medium.
3rd, the purifying of Bashan Mountain Chinese torreya endophyte of plant
When inoculum tangent plane edge grows tiny mycelia in plate, consolidated with the new PDA for preparing of transfer needle picking mycelia implantation
On body flat board, purified repeatedly in PDA culture medium, until obtaining pure culture.
Embodiment 2：Endogenetic fungus bacteriostatic activity is detected
1st, after will be for examination bacterium (staphylococcus aureus, Escherichia coli) LB fluid nutrient mediums activation, it be respectively coated on
LB solid culture primary surfaces；
2nd, the purifying endophyte of plant agar medium block of the acquisition of embodiment 1 is cut, places be inoculated in golden yellow Portugal respectively
In grape coccus and Escherichia coli plate, 37 DEG C are cultivated 1-2 days, the inhibition zone around observation agar block.
By the above method, screening obtains one plant has anti-Staphylococcus aureus (G simultaneously+) and Escherichia coli (G-) living
The bacterial strain of property, is designated as BP6T2.
Embodiment 3：Endogenetic fungus BP6T2 identification
Observed by plate cultural character, the bacterial strain colony colour is in grass green, bacterium colony quality is velvet (Fig. 2)；It is micro-
Microscopic observation conidial head is in loose radial, and top capsule is subsphaeroidal, and stigma individual layer is double-deck to have (Fig. 3) simultaneously, according to《China
Fungi will aspergillus of volume five and its related epigamous》(Qi Zutong is edited, Chinese fungi will (volume five) aspergillus and its correlation
Epigamous [M] Beijing：Science Press, 1997,5~aspergillus flavus (Aspergillus flavus) 10.) can be initially identified as
BP6T2。
Embodiment 4：The extraction of liposoluble substance in aspergillus flavus BP6T2 zymotic fluids
1st, the aspergillus flavus BP6T2 that embodiment 2 is obtained is activated into (activation condition in PDA liquid medium：28 DEG C,
180rpm, is cultivated 5-7 days) it is used as seed liquor；
2nd, accessed according to 5% (v/v) inoculum concentration in PDA liquid medium, 28 DEG C, 180rpm is cultivated 7-10 days；
3rd, the ethyl acetate that 1/3 volume is added in filtrate, filtrate is collected by filtration, counter-current extraction 3 times collects upper strata organic
Phase；
4th, organic phase dissolves material and the volatilization being bonded on wall with methanol in 35 DEG C of removing organic solvents on Rotary Evaporators
Dry, pass through gravimetric detemination zymotic fluid fat-soluble extract content；Determine fat-soluble extract in aspergillus flavus BP6T2 zymotic fluids
Content is averagely about 93mg/L.
Embodiment 5：High performance liquid chromatography (HPLC) quantitatively detects aspergillus flavus BP6T2 extract paclitaxel analogs
1st, the fat-soluble extract and flowing phase processor obtained in embodiment 4：The enriched product that will be obtained in embodiment 4
13200r/min centrifuges 2min, and Aspirate supernatant removes impurity, then with the mobile phase prepared through 0.22 μm of aperture membrane filtration
(methanol/water=65/35, V/V) ultrasonic wave is vented together, and the time is respectively 3min, 10~15min；
2nd, chromatographic condition is：Chromatographic column, ODS (C18) posts 4.6 × 250mm, 5nm；Column temperature, normal temperature；Sample and mobile phase,
Methanol/water=65/35 (V/V)；Flow velocity, 1.0mL/min；Sample injection volume, 20 μ L；Ultraviolet detection wavelength, 227nm；
3rd, the Rt (retention time) for measuring Taxol Standard (Sigam) is 13~14min (Fig. 4)；
4th, in extract sample made from embodiment 4 find with Taxol Standard retention time it is basically identical into
Point, appearance time is 13.208min (Fig. 5), can be initially identified as paclitaxel analogs；
5th, quantitative analysis：Aspergillus flavus BP6T2 is calculated according to chromatogram using the rich liquid chromatogram workstation softwares of HPLC 5
The relative peak area for being metabolized paclitaxel analogs in extract is 2491.2 (Fig. 5), and percentage contents are averagely about 8.4%.
Embodiment 6：LC-MS instrument (HPLC-MS) determines that aspergillus flavus BP6T2 zymotic fluids contain taxol
1st, liquid chromatogram INSTRUMENT MODEL：Shimadzu LC-30A UPLC
(1) sample and flowing phase processor：Fat-soluble extract prepared by embodiment 4,13200r/min centrifugation 2min, inhales
Supernatant is taken, impurity is removed through 0.22 μm of aperture membrane filtration, then with the mobile phase (acetonitrile/water=65/35, V/V) prepared
Ultrasonic wave is vented together, and the time is respectively 3min, 10~15min；
(2) chromatographic condition is：Chromatographic column, ODS (C18) post 4.6x 250mm, 5nm；Column temperature, normal temperature；Sample and mobile phase,
Methanol/water=65/35 (V/V)；Flow velocity, 1.0mL/min；Sample injection volume, 20 μ L；Ultraviolet detection wavelength, 227nm；
2nd, mass spectrometer model AB sciex 4500QQQ (triple level Four bar LC-MS instrument)
The structure (table 1) of paclitaxel analogs in metabolism extract is determined by LC-MS instrument, shows its parent ion Da
For 876.300, daughter ion Da is respectively 307.900,591.200 and 531.200, and source voltage is 150, and impact energy is respectively
36th, 36 and 40, it is completely the same with bearing taxanes Taxol Standard, can be by taxol in aspergillus flavus BP6T2 zymotic fluids
Qualitative analog is taxol.
The mass spectral analysis parameter of the Taxol Standard of table 1 and paclitaxel analogs in aspergillus flavus BP6T2 zymotic fluids
| Q1Mass(Da) | Q3Mass(Da) | DP | CE | Dwell(msec) |
| 876.300 | 307.900 | 150 | 36 | 50 |
| 876.300 | 591.200 | 150 | 36 | 50 |
| 876.300 | 531.200 | 150 | 40 | 50 |
Embodiment 7：Inhibitory action of the aspergillus flavus BP6T2 fermentation broth extracts to Hela tumour cells
1st, take the logarithm Hela cells (Hua Lian Bioisystech Co., Ltd of the section) bed board in growth period, cell is pressed into 100 μ L/ holes
(1~5 × 103Individual cells/well) it is inoculated in 96 orifice plates, it is placed in 37 DEG C, 5%CO224h is cultivated in incubator；
2nd, the aspergillus flavus BP6T2 fermentation broth extracts containing taxol in embodiment 4 are configured into various concentrations (table 2), takes 10 μ
L is separately added into sample well, 37 DEG C, 5%CO224h is cultivated in incubator；
3rd, culture plate is taken out, supernatant is sucked, (the DMEM trainings containing 10% hyclone of 90 μ L fresh mediums are added per hole
Nutrient solution) and μ L of CCK-8 10, it is same under conditions of continue to cultivate 4h；
4th, A is read at 450nm with ELIASA450Value, calculates inhibitory rate of cell growth, and inhibitory rate of cell growth is calculated：
Hela cell inhibitory rates (IR)=(control wells A values-sample well A values)/control wells A value × 100%.
Pass through above-mentioned experiment, it was demonstrated that aspergillus flavus BP6T2 fermentation broth extracts have obvious inhibitory action (table to tumour cell
2) rise of the concentration, and with extract, the inhibitory action enhancing of cell proliferation, wherein being handled with 3.104 μ g/mL concentration
Inhibitory action it is most strong, inhibiting rate is 66.20%.When minimum concentration is 0.0310 μ g/mL, inhibiting rate is 39.90%, therefore is pushed away
The anti-Hela cellular materials for surveying aspergillus flavus BP6T2 generations are taxol.
Inhibitory action of the aspergillus flavus BP6T2 fermentation broth extracts of table 2 to Hela cells
| Sample concentration (μ g/mL) | A450Value | Inhibiting rate (%) |
| Blank control | 1.153 | ---- |
| 0.0310 | 0.693 | 39.9% |
| 0.3104 | 0.615 | 46.7% |
| 3.104 | 0.39 | 66.2% |
Claims (5)
1. one plant of paclitaxel produced endophyte, it is characterised in that：Described endophyte is aspergillus flavus BP6T2, and deposit number is
CCTCC NO：M2016318.
2. endophyte according to claim 1, it is characterised in that：Described endophyte has paclitaxel produced characteristic, hair
The content of taxol reaches 7.81mg/L in zymotic fluid.
3. endophyte according to claim 1, it is characterised in that：Described endophyte is divided from the tissue of Bashan Mountain Chinese torreya
Separate out what is come.
4. endophyte according to claim 1, it is characterised in that：Described endophyte have anti-Staphylococcus aureus and
The activity of Escherichia coli.
5. application of the endophyte in treatment tumour cell medicine is prepared described in claim 1, described tumour cell is palace
Neck cancer cell.
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| CN111719014A (en) * | 2020-07-21 | 2020-09-29 | 中南大学 | Method for rapidly identifying plant flavonoid-producing endophyte |
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