CN108102928B - One plant of gingko endogenous fungus and its application - Google Patents
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Abstract
The invention discloses one plant of gingko endogenous fungus and its applications.The present invention is isolated to the raw sickle-like bacteria of gingko endogenous fungus layer (Fusarium proliferatum) DZHQ1 with anti-tumor activity from ginkgo bark, the method combined is sequenced by colonial morphology and 18sRNA, judge the kind of bacterial strain, then the anti-cervical cancer activity of mtt assay detection bacterial strain crude extract is utilized, the method for finally preparing HPLC using half, the secondary metabolite of bacterial strain to inhibiting rate greater than 50% separates, and helps further to screen compound monomer with anti-tumor activity.
Description
Technical field
The invention belongs to microorganism field and therapeutic field of tumor, and in particular to one plant of gingko endogenous fungus and its anti-swollen
Application in terms of tumor.
Background technique
Cancer is to endanger one of the principal disease of human health now, seriously threatens the life of the mankind.Cervical carcinoma is most
Common gynecologic malignant tumor, its morbidity has the tendency that rejuvenation in recent years.Current clinically common anti-tumor drug has 50
It is a variety of, but most drugs can only make disease amelioration, be unable to reach the purpose cured completely.So the research and development one of anti-cancer agent
It is directly the main aspect in drug research field.
Ginkgo is China's specialty medicinal plant, so far it has been found that ginkgo leaf contains more than 100 kinds of chemical components, mainly there is Huang
Ketone compounds, terpene lactones compound, bilobetin, polypentenol etc. all have antitumor action in extract.It is common
Ginkgo biloba extract mainly has ginkgo leaf composite extract (EGb), Polyprenols From Ginkgo Biloba L (GP), Ginkgo biloba polysaccharide class compound
(GBLP), four major class of gingko exocarp polysaccharide (GBEP).Contain Multiple components, mainly flavone compound and terpenes in EGb,
Research find EGb to mouse transplanted sarcoma S180 and Murine Hepatoma22 cell in vivo, outer have inhibiting effect (Ji Yufeng etc., ginkgo
Experimental study TCM investigation .2005,18 (7): 14-16 of extract antitumor action).GP is in vitro for gastric carcinoma cells
Strain SGC-7901, human colon adenocarcinoma cell strain LOVO and human cervical carcinoma Hela cell's strain inhibitory rate 60%~80%, in vivo
To the inhibitory rate of transplanted tumor cell Heps, S180 and EC to 50%~65% (Wang Chengzhang etc., ginkgo leaf polypren phosphorus
The antitumor bioactivity research chemistry of forest product in acid esters inside and outside and industry .2006,26 (3): 13-16).GBEP is 10~320
Under μ g/mL dosage, 24~72h of interaction in vitro, to BEL-7404 human hepatocellular cells, Wei-wei Decoction SGC-7901 and lung gland
Cancer cell line SPC-A-1 all has inhibiting effect.The antitumor possibility mechanism of action of ginkgo biloba extract has: anti-oxidant and removing is freely
The adjusting of tumour and related gene is made in base, the proliferation for influencing tumour cell and apoptosis-induced, the tumor vascular formation of inhibition
With, to cytotoxicity of tumour cell etc..
However Ginkgo Growing is slow, takes 20 years or more from being planted to result under natural conditions, constrains its medicinal efficacy
Exploitation infers that it is very likely that there is certain endogenetic fungus in ginkgo according to plant endogenesis epiphyte endosymbiotic theory, can generate
With the same or similar chemical component of ginkgo, so gingko endogenous fungus has become a hot topic of research, people expect that it can become silver
The new sources of apricot class drug or the new way of its bioactive substance.And the research about gingko endogenous fungus is fewer, especially
It is research for gingko endogenous fungus anti-tumor activity is even more few few.Guo Jian is newly equal to be separated from the ginkgo of Yangling Shaanxi
522 plants of endogenetic fungus are obtained, its inhibiting effect to 7 kinds of plant pathogenic fungis are determined using mycelia growth inhibition assay, as a result
Showing the bacterial strain for accounting for sum 50.7% has antibacterial activity (Guo Jian is new etc., point of Antifungal Isolates from Endophytic Fungi in Ginkgo Biloba L
From with screening the northwest agricultural journal .2005,14 (4): 14-17).The antitumor activity in vitro such as Miao Li show Fuyang area
19 plants of isolated gingko endogenous fungus, wherein active strongest bacterial strain is YX5, fermentation broth coarse extract is to tumour cell
The inhibiting rate IC of EC109, human nasopharyngeal carcinoma HONE1 and human cervical carcinoma HeLa50Respectively 18.3,3.6 and 6.5 μ g/ml (Miao Li, king
First, Zhu Lei, Wu Zhengjun, the separation of Zhou Rumei Four Plants endogenetic fungus and its screening microbiology of anti-tumor activity are logical
Report .2009,36 (6): 865-869).
Carry out Gingko biloba plant endogenetic fungus active matter Quality Research, to the exploitation medicinal endogenetic fungus resource of China's Gingko biloba plant
And new microbial medicine all has important theory significance and potential application value.
Summary of the invention
In view of the above-mentioned problems of the prior art, the present invention is studied for gingko endogenous fungus, it is anti-to obtain
The drug or bioactive substance of tumour.
For achieving the above object, specifically, the present invention relates to following technical schemes:
Firstly, the invention discloses one plant of raw sickle-like bacteria of gingko endogenous fungus layer (Fusarium proliferatum)
DZHQ1, deposit number are CGMCC No.14983, are preserved in Chinese microorganism strain preservation management on November 28th, 2017
Committee's common micro-organisms center.
Secondly, can be activated, be cultivated in PDA culture the invention discloses the cultural method of the endogenetic fungus
Or fermentation.
Further, the invention discloses the endogenetic fungus to prepare the application in antitumor active medicine.It is preferred that
, the tumour is cervical carcinoma.
In addition, the invention also discloses the extractions of the ethyl acetate of the fermentation culture of the endogenetic fungus, fermentation culture
Object all has anti-tumor activity, has therapeutic activity in particular for cervical carcinoma.
The invention also discloses a kind of anti-tumor activity compositions, and it includes the fermentation cultures of the endogenetic fungus, hair
The ethyl acetate extract of ferment culture solution.
Preferably, above-mentioned composition can further include pharmaceutically acceptable carrier.
Described carrier refers to the carrier of pharmaceutical field routine, such as diluent, dispersing agent, stabilizer.
The present invention achieve it is following the utility model has the advantages that
The present invention is isolated to the active raw sickle-like bacteria of gingko endogenous fungus layer of anti-cervical cancer from ginkgo bark
(Fusarium proliferatum) DZHQ1 is sequenced the method combined by colonial morphology and 18sRNA, judges bacterial strain
Then kind utilizes the anti-cervical cancer activity of mtt assay detection bacterial strain crude extract, the method for finally preparing HPLC using half, to suppression
The secondary metabolite of bacterial strain of the rate processed greater than 50% is separated, and helps further to screen chemical combination with anti-tumor activity
Object monomer.
Detailed description of the invention
The bacterium colony and hypha form of tri- plants of gingko endogenous fungus of Fig. 1, a bacterial strain J-1；B bacterial strain J-2；C bacterial strain J-3；
The systematic evolution tree 1 of tri- plants of gingko endogenous fungus of Fig. 2: bacterial strain J-1；2: bacterial strain J-2；3: bacterial strain J-3；
The active MTT detection of Fig. 3 bacterial strain J-1 and J-3 anti-cervical cancer (processing is for 24 hours)
The HPLC peak shape figure of Fig. 4 bacterial strain J-1 secondary metabolite
The HPLC peak shape figure of Fig. 5 bacterial strain J-3 secondary metabolite
Specific embodiment
The present invention will be further explained with reference to the examples below, as described below, is only to preferable implementation of the invention
Example, not limits the present invention, any person skilled in the art is possibly also with the disclosure above
Technology contents be changed to the equivalent embodiment changed on an equal basis.Without departing from the concept of the present invention, according to the present invention
Technical spirit any simple modification or equivalent variations that following embodiment is made, fall within the scope of protection of the present invention.
Embodiment 1: the screening and identification of gingko endogenous fungus
Separation material: ginkgo picks up from Shandong Linyi City, and materials position is the trunk barks away from ground 2cm or so, and thickness is about
3-8mm, the ginkgo age of tree 30 years or so, trunk diameter about 55cm.The fresh ginkgo bark for taking acquisition is washed its surface with distilled water
Completely, exocuticle is removed after slightly dry, the segment for being cut into suitable length in its xylem is taken, 6min is impregnated in 75% ethyl alcohol, then use
Aseptic water washing 2-3 times, aseptic filter paper blot.Then it is cut into fritter and is put in the PDA culture medium containing streptomysin and penicillin
On, it is placed in 25 DEG C of incubators and cultivates 3-20d, after growing mycelia around organizing, transfer them in new PDA culture medium,
Continue to cultivate in 25 DEG C of incubators.Until the colony identification grown is pure bacterium.Sterile glycerol pipe of the bacterial strain after purification 10%
Interior -80 DEG C of preservations.The endogenetic fungus (respectively J-1, J-2, J-3 are naming certainly for inventor) for obtaining three plants of purifying is isolated,
Every plant of bacterium takes 10ml fermentation liquid, send to Bo Shang biotechnology (Shanghai) Co., Ltd. and carries out 18sRNA sequencing analysis.
Three plants of endogenetic fungus colonial morphologies are as shown in Figure 1, the systematic evolution tree of three plants of gingko endogenous fungus is as shown in Figure 2.
The 18sRNA of bacterial strain J-1 and J-2 and the similarity of the raw sickle-like bacteria of layer are respectively 99% and 98%, in combination with the knot of colony characteristics
It is raw Fusarium that fruit shows that bacterial strain J-1 and J-2 belong to layer (see Fig. 1 and Fig. 2).And the 18sRNA sequence of J-3 and the raw sickle-like bacteria of layer
The conserved sequence similarity of category is only 97%, therefore initial guess bacterial strain J-3 may also belong to Fusarium, but is needed further
Spore shape is observed to confirm even with other molecular biology methods.
Embodiment 2: the antitumor activity of gingko endogenous fungus
The preparation of fermentation liquid and mycelia crude extract: taking cultured three plants of gingko endogenous fungus in PDA solid medium,
By inoculated by hypha block in the triangular flask equipped with 250ml PDA liquid medium, 20 DEG C, 120r/min shaking table culture 7d.Then
Ethyl acetate is added by the volume ratio of 1:1 into each culture bottle, 4d is cultivated in continuation in shaking table.Fermentation liquid is through 3-4 layers of gauze
Mycelium is removed after filtering, is layered with separatory funnel, and the organic phase containing gingko endogenous fungus secondary metabolite is obtained.Benefit
Ethyl acetate most of in organic phase are recycled with Rotary Evaporators, are obtained containing the dense of gingko endogenous fungus secondary metabolite
Contracting liquid.Finally, being completely dried concentrate using vacuum concentration drier, being dissolved in is thallus crude extract in DMSO.
Mtt assay detects the anti-cervical cancer activity of crude extract: Cervical Cancer HeLa Cells system used, the culture medium of cell is cultivated:
10% newborn bovine serum, 89% RPMI1640 complete medium, 1% dual anti-(Pen .- Strep) lead at 37 DEG C
Enter 5%CO2Cell incubator in subculture, using mtt assay measurement crude extract anti-cervical cancer tumor promotion.Take logarithm raw
Cell suspending liquid is made with complete medium after trypsin digestion in long-term tumour cell, and blood counting chamber counts, with
After take 100 μ L to be inoculated in 96 orifice plates, reserve two holes as blank control.It is placed in 5%CO2In incubator after 37 DEG C of culture 48h, add
Enter 20 μ L through the diluted sample of complete culture solution (negative control group adds 20 μ L culture solutions, and the culture solution of 100 μ L is added in blank group,
Each sample does 3 repetitions), continue to take out after cultivating 2d, draws culture medium in hole and lose, the MTT of 2.5 μ g/ μ L is added in every hole
Solution 20 μ L, after reacting 4h at 37 DEG C, every Kong Zaijia 100 μ L DMSO dissolves 30min at 37 DEG C；Each hole is measured with microplate reader
Light absorption value (measurement wavelength distinguish 570nm).Inhibiting rate is finally calculated as follows: inhibiting rate=(negative control OD value-experimental group
OD value)/(negative control OD value-blank control OD value) × 100%.
Fermentation broth coarse extract anti-cervical cancer determination of activity result (Fig. 3) display, bacterial strain J-1 and J-3 are to cervical cancer cell
Inhibiting rate is respectively 59.6% and 59.1%, and the inhibiting rate of J-2 is only 13.3%.Therefore selection bacterial strain J-1 and J-3 is carried out down
The experiment of one step.
It chooses the higher J-1 bacterial strain of anti-cervical cancer activity and carries out preservation, the raw sickle-like bacteria (Fusarium of the entitled layer of preservation
Proliferatum) DZHQ1, deposit number are CGMCC No.14983, have been preserved in the micro- life of China on November 28th, 2017
Object culture presevation administration committee common micro-organisms center (abbreviation CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number, Institute of Microorganism, Academia Sinica, postcode 100101).
3 gingko endogenous fungus anti-tumor active substance research of embodiment
Half preparation HPLC method Analyze & separate gingko endogenous fungus secondary metabolite: chromatographic column is C18 column
(250mmx10mm, 5 μm), mobile phase is methanol-water.The elution of 20% methanol, volume flow 1.0ml/min, Detection wavelength
210nm, 35 DEG C of column temperature, 100 μ l of sample volume.According to the peak shape of different strains secondary metabolite, different substances is isolated.Again
Each substance and Cervical Cancer HeLa Cells are co-cultured, determine effective anti-cervical cancer tumor promotion substance using mtt assay.
HPLC method being prepared using half, separation determination being carried out to the secondary metabolite of bacterial strain J-1 and J-3, separating resulting is as schemed
Shown in 4 and Fig. 5, the secondary metabolite as can be seen that bacterial strain J-1 and J-3 is prepared on HPLC peak shape figure (Fig. 4 and Fig. 5) from half
Difference, and all containing there are many kinds of substances.The compound of each peak shape representative is collected, carrying out MTT detection to determine has anti-palace
The monomeric compound of neck cancer tumor promotion.
Claims (5)
1. one plant of raw sickle-like bacteria of gingko endogenous fungus layer (Fusarium proliferatum) DZHQ1, deposit number CGMCC
No.14983 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on November 28th, 2017.
2. a kind of fermentation culture of the raw sickle-like bacteria of gingko endogenous fungus layer as described in claim 1, which is characterized in that preparation
Method is as follows: after the endogenetic fungus is inoculated in fluid nutrient medium seven days, resulting liquid medium is the endophyte
Fermentation culture.
3. a kind of ethyl acetate of fermentation culture of the raw sickle-like bacteria of gingko endogenous fungus layer as described in claim 1 extracts
Object, which is characterized in that the preparation method is as follows:
After inoculated by hypha block is cultivated seven days after fluid nutrient medium, ethyl acetate training is added into culture medium with volume ratio 1:1
It supports four days, after being filtered to remove mycelia, recycles containing the ethyl acetate extract in gingko endogenous fungus secondary metabolite organic phase,
The ethyl acetate extract that substance obtained in DMSO is fermentation culture is dissolved in after ethyl acetate extract is concentrated and dried.
4. a kind of anti-cervical cancer active compound, which is characterized in that wanted comprising fermentation culture as claimed in claim 2 and right
The ethyl acetate extract of fermentation culture described in asking 3.
5. composition according to claim 4, which is characterized in that the composition includes pharmaceutically acceptable load
Body；The carrier is selected from one or more of diluent, dispersing agent, stabilizer.
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| CN201711396247.XA CN108102928B (en) | 2017-12-21 | 2017-12-21 | One plant of gingko endogenous fungus and its application |
| PCT/CN2018/113709 WO2019119992A1 (en) | 2017-12-21 | 2018-11-02 | Endophytic fungus of ginkgo biloba and metabolite product and application thereof |
| US16/469,830 US11332707B2 (en) | 2017-12-21 | 2018-11-02 | Endophytic fungus from gingko, metabolite product and use thereof |
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| CN109082445B (en) * | 2018-08-30 | 2020-06-05 | 德州学院 | Metabolite product of ginkgo endophytic fungi and application of metabolite product in antibiosis |
| WO2019119992A1 (en) * | 2017-12-21 | 2019-06-27 | 德州学院 | Endophytic fungus of ginkgo biloba and metabolite product and application thereof |
| CN116419976A (en) * | 2021-03-12 | 2023-07-11 | 德州学院 | Ginkgo endophytic fungus and application thereof in resisting bacteria and tumors |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676398A (en) * | 2012-03-14 | 2012-09-19 | 安徽农业大学 | Separation and purification method of endophytic fungi from ginkgo biloba |
| KR20120121076A (en) * | 2011-04-26 | 2012-11-05 | (주)아모레퍼시픽 | Method of producing Ginsenoside F1 from the Ginsenoside Rg1 by extracellular enzyme from Fusarium moniliforme var. subglutinans |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20120121076A (en) * | 2011-04-26 | 2012-11-05 | (주)아모레퍼시픽 | Method of producing Ginsenoside F1 from the Ginsenoside Rg1 by extracellular enzyme from Fusarium moniliforme var. subglutinans |
| CN102676398A (en) * | 2012-03-14 | 2012-09-19 | 安徽农业大学 | Separation and purification method of endophytic fungi from ginkgo biloba |
Non-Patent Citations (2)
| Title |
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| Antifungal Attributes of Lactobacillus plantarum MYS6 against Fumonisin Producing Fusarium proliferatum Associated with Poultry Feeds;B. V. Deepthi等;《PLOS ONE》;20160610;第1-22页，参见全文 * |
| 四种植物内生真菌的分离及其抗肿瘤活性的筛选;缪莉等;《微生物学通报》;20090620;第36卷(第6期);第865-869页，参见"材料与方法" * |
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