CN110092794A - The method and application of chaetomium globosum QY-1 separation and Extraction chaetomium globosum A - Google Patents
The method and application of chaetomium globosum QY-1 separation and Extraction chaetomium globosum A Download PDFInfo
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- CN110092794A CN110092794A CN201910407835.1A CN201910407835A CN110092794A CN 110092794 A CN110092794 A CN 110092794A CN 201910407835 A CN201910407835 A CN 201910407835A CN 110092794 A CN110092794 A CN 110092794A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
- C07D491/10—Spiro-condensed systems
- C07D491/107—Spiro-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
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Abstract
The invention discloses chaetomium globosum QY-1(Chaetomium globosum) product chaetomium globosum A separating and extracting process and application.The present invention is using chaetomium globosum QY-1 as raw material, and by fermented and cultured, vacuum filtration, ethyl acetate extraction through silica gel column chromatography acetone-petroleum ether gradient elution, liquid phase analysis and preparation, crystallizes, is recrystallized to give secondary metabolite.This secondary metabolite is total by nuclear-magnetism1H‑NMR、13It is 528.6 that tri- spectrograms of C-NMR, DEPT, which are accredited as chaetomium globosum A molecular weight, molecular formula C32H36N2O5, shape is in pale yellow powder shape.The experiment proved that, chaetomium globosum A has good antioxidant activity, and it is also relatively high in the content of fermentation liquid, it can be used for the preparation of natural, antioxidant to develop novel provides selection, it can be applied to the fields such as food, medicine and cosmetics, there is very high researching value and good economic and social benefit.
Description
Technical field
The invention discloses the separating and extracting process of the product chaetomium globosum A of chaetomium globosum QY-1 and applications.It can be used for
Oxidation resistant application, belongs to biomedicine technical field.
Background technique
Chaetomium globosum (Chaetomium globosum) belongs to Ascomycetes (Ascomycetes) spherical shell bacterium
(Spliacrialcs) cupreum section (Chaetomiaceae) Chaetomium (Chaetomiiun) is widely present in soil, plants
It is ripe by people institute due to generating chaetomium globosum (chaetoglobosins) on object, clast and cellulose-containing product
Know.
Chaetomium globosum QY-1 is isolated from a kind of endogenetic fungus of the leaf portion of M. laxiflora before the Riparian Zone waterflooding of Three Gorges.Fungi
Number of types of secondary metabolite can be generated during the growth process, it is very close with human relation, meanwhile, cometabolism produces
Object plays the role of critically important in fields such as natural drug, cosmetics, such as broad-spectrum medicinal such as cephalosporin, ergotin are all true
The natural products of bacterium.
The secondary metabolite of chaetomium globosum is very rich, including alkaloids, thermophilic azone class, terpene, esters, flavones
Class, xanthone etc., wherein the compound of alkaloids accounts for major part, including ball chaetocin class compound, diketopiperazines
Deng.So chaetomium globosum has many bioactivity, such as antitumor, antibacterial, anti-inflammatory, antiviral isoreactivity.
Summary of the invention
An object of the present invention is to provide a kind of chaetomium globosum (Chaetomium globosum) QY-1 bacterial strain secondary generation
Thank to the isolation and identification method of product chaetomium globosum A, however easily isomerization occurs for chaetomium globosum A and unstable and less soluble
Yu Shui is soluble in the organic solvents such as methanol, ethyl alcohol.The secondary metabolite chaetomium globosum A of the chaetomium globosum has good
Antioxidation, and the content of its fermentation liquid chaetomium globosum A is also relatively high, can be used for preparing antioxidant, it is new to develop
Antioxidant provide new selection.
The method of chaetomium globosum QY-1 separation and Extraction chaetomium globosum A, includes the following steps: to consolidate QY-1 strain in PDA
In body culture medium, 3-7d is cultivated, then mycelia will be inoculated in PDA liquid medium, 25-28 DEG C, 120-140r/min culture 5-7d
Gained.Fermented and cultured product, which is filtered by vacuum, mutually separates fermentation liquid water phase with thallus, the bodies such as fermentation liquid water phase and ethyl acetate
Three times, combining extraction liquid, decompression low temperature is concentrated to get chaetomium globosum QY-1 ethyl acetate phase crude extract, chaetomium globosum for product extraction
QY-1 ester phase crude extract crystallizes through silica gel column chromatography acetone-petroleum ether gradient elution, is recrystallized to give chaetomium globosum A, this
Grade metabolite determines structural formula by nuclear magnetic resonance are as follows:
The chaetomium globosum QY-1 is preserved in China typical culture collection center, classification life on April 7th, 2016
Name Chaetomium globosum, deposit number CCTCC NO:M 2016178.
Another technical solution of the invention is using the isolated chaetomium globosum A as the application of antioxidant.
Oxidation resistant effect of the present invention shows the secondary metabolite chaetomium globosum A's of chaetomium globosum QY-1
Crude extract DPPH free radical scavenging activity is 52.81%, and total antioxidation is 8.75 units/mL, the object of chaetomium globosum A in fermentation liquid
Matter content accounts for about the 52.20% of total material content.Thus, the invention also includes the biologies containing the bacterial strain or its fermentation liquid
Pesticide, and the microbial bacterial agent containing the bacterial strain.
Detailed description of the invention
Fig. 1 is the secondary metabolite separation and Extraction of chaetomium globosum (Chaetomium globosum) QY-1 of the invention
Flow chart.
Fig. 2 is the secondary metabolite ethyl acetate phase liquid phase figure of QY-1.
Fig. 3 is the secondary metabolite chaetomium globosum A liquid phase figure of QY-1.
Fig. 4 is the secondary metabolite chaetomium globosum A's of QY-11H-NMR spectrum.
Fig. 5 is the secondary metabolite chaetomium globosum A's of QY-113C-NMR spectrogram.
Fig. 6 is the DEPT spectrogram of the secondary metabolite chaetomium globosum A of QY-1.
Fig. 7 is the canonical plotting of chaetomium globosum A assay.
Specific embodiment
Embodiment 1: the separation and Extraction of the secondary metabolite of chaetomium globosum (Chaetomium globosum) QY-1, mirror
It is fixed
The activation and culture of strain
The QY-1 strain that picking saves cultivates 3-7d in PDA solid medium, is inoculated with mycelia in fermentation medium (PDA
Fluid nutrient medium), 500mL triangular flask liquid amount is 200mL, 28 DEG C, 140r/min culture 7d.
Sample extraction
The bacteria suspension fermented is removed by suction filtration under vacuum mycelia and obtains fermentation liquid water phase, the isometric acetic acid of fermentation liquid water phase
Ethyl ester extracts three times, and combining extraction liquid obtains the acetic acid second phase of the fermentation liquid of the bacterial strain secondary metabolite, is then concentrated under reduced pressure,
It is freeze-dried spare, as QY-1 fermentation liquid ethyl acetate phase crude extract.
The separation of substance is identified
The determination of eluant, eluent: before carrying out silica gel column chromatography, it is necessary first to determine a kind of eluant, eluent of good separating effect.
At identical conditions, silica gel column chromatography and thin-layer chromatography have something in common in the principle of separate substance, therefore in selection silicon
When the eluent of plastic column chromatography, is first screened with easy thin-layer chromatography, select several common solvents to be tested, according to Rf
(0.3 < Rf < 0.7) value selects suitable solvent.The suitable solvent of QY-1 fermentation liquid ethyl acetate phase crude extract of the present invention is
Acetone/petroleum ether.
Silica gel column chromatography: taking fermentation liquid ethyl acetate phase crude extract to be dissolved in methanol, and suitable silica white (200-300 is added
Mesh) it mixes, to loading after low temperature drying.Column silica gel for chromatography powder (200-300 mesh) will be taken to activate 30min at 105 DEG C, shifted
It is cooling into drier.Using dry column-packing and loading, i.e., directly add activated silica white in pillar, holding is put down above
It is whole, the silica white for having mixed sample is added later, is finally eluted with different proportion acetone/petroleum ether eluant, eluent, collection evaporates
Point, it is to be analyzed to dry up sample for vacuum distillation.
The high performance liquid chromatography of fraction: HPLC detection is carried out to each fraction of crude extract, passes through eluting peak each in sample
The purity of retention time judgement sample collects the fraction of single envelope compound.Secondary metabolite of the invention, liquid phase are prepared molten
Liquid drying organic phase has yellow crystal, and (4 DEG C) of low temperature recrystallizations are separated by filtration crystallization and mother liquor, collect crystal.
Nmr analysis: the compound of crystal is obtained by collecting, nmr analysis is carried out, determines its structure.
It is analyzed by Structural Identification, obtaining a pure compound is chaetomium globosum A, and structure is as follows:
Embodiment 2: the assay of chaetomium globosum A
Chaetomium globosum A is dissolved into the solution of various concentration gradient with methanol (chromatographic grade), it is identical when being prepared with separation
Liquid-phase condition analyzed, record various concentration chaetomium globosum solution A peak area, make standard curve.First is used later
Alcohol dissolves a small amount of crude extract, and is analyzed with liquid-phase condition identical when separating preparation, record liquid phase peak position
Then the formula that peak area is brought into standard curve is calculated the content of principal goods matter CheA in crude extract, standard by peak area
Shown in the Dependence Results such as following figure (Fig. 7).
In crude extract shown in the content results of substance such as following table (table 1):
The content results of 1 substance of table
The result shows that if condition of culture is suitable for, the amount that QY-1 bacterial strain produces principal goods matter CheA can achieve 379.76 ±
37.49mg/L is that the QY-1 bacterial strain reported at present produces seven times of amount 53.71mg/L or so of CheA.
Embodiment 3: the antioxidant activity of the secondary metabolite of chaetomium globosum (Chaetomium globosum) QY-1 point
Analysis
Antioxidant activity analysis for the secondary metabolite fermentation liquid ethyl acetate phase of QY-1:
DPPH method, T-AOC method are mainly used, the potassium ferricyanide restores force method and measures antioxidant activity, sample concentration 10mg/
ML, positive control are the Vc aqueous solution with concentration.
As a result shown in following (table 2):
The antioxidant activity of the secondary metabolite fermentation liquid ethyl acetate phase of 2 QY-1 of table is analyzed
Antioxidant activity analysis for the secondary metabolite chaetomium globosum A of QY-1:
DPPH method, T-AOC method are mainly used, the potassium ferricyanide restores force method and measures antioxidant activity, sample concentration 1mg/
ML, positive control are the Vc aqueous solution with concentration.As a result as shown in following table (table 3):
The antioxidant activity of 3 chaetomium globosum A of table is analyzed
Claims (5)
1. the method for chaetomium globosum QY-1 separation and Extraction chaetomium globosum A, which comprises the steps of: by raw material ball
Cupreum QY-1 fermented and cultured, fermented and cultured product, which is filtered by vacuum, mutually separates fermentation liquid water phase with thallus, fermentation liquid water phase
Three times with ethyl acetate equal-volume extraction, combining extraction liquid, it is thick that decompression low temperature is concentrated to get chaetomium globosum QY-1 ethyl acetate phase
Extract, chaetomium globosum QY-1 ester phase crude extract crystallize through silica gel column chromatography acetone-petroleum ether gradient elution, are recrystallized to give ball
Chaetomin A, the structural formula of chaetomium globosum A are as follows:
2. the method for chaetomium globosum QY-1 separation and Extraction chaetomium globosum A according to claim 1, which is characterized in that ball
Cupreum QY-1 fermented and cultured process be by QY-1 strain in PDA solid medium, cultivate 3-7d, then will inoculation mycelia in
PDA liquid medium, 25-28 DEG C, 120-140r/min culture 5-7d gained.
3. the method for chaetomium globosum QY-1 separation and Extraction chaetomium globosum A according to claim 1, which is characterized in that institute
The chaetomium globosum QY-1 stated is preserved in China typical culture collection center, classification naming on April 7th, 2016
Chaetomium globosum, deposit number CCTCC NO:M 2016178.
4. isolated chaetomium globosum QY-1 ethyl acetate phase crude extract answering as antioxidant according to claim 1
With.
5. application of any one of -3 isolated chaetomium globosum A as antioxidant according to claim 1.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110169447A (en) * | 2019-05-15 | 2019-08-27 | 三峡大学 | Chaetomium globosum A causes the application on rotten bacterium medicament as citrus |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102754652A (en) * | 2012-06-06 | 2012-10-31 | 吉林大学 | New purpose of chaetoglobosin A |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102754652A (en) * | 2012-06-06 | 2012-10-31 | 吉林大学 | New purpose of chaetoglobosin A |
Non-Patent Citations (3)
| Title |
|---|
| CHENG JIANG等: "New production process of chaetoglobosin A using cornstalksthe antifungal", 《BRAZILIANJOURNAL OF MICROBIOLOGY》 * |
| LI, WEI等: "Anti-phytopathogen, multi-target acetylcholinesterase inhibitory and antioxidant activities of metabolites from endophytic Chaetomium globosum", 《NATURAL PRODUCT RESEARCH》 * |
| 张文豪等: "侧柏内生真菌Chaetomium globosum ZH-32抗菌活性成分研究", 《农药学学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110169447A (en) * | 2019-05-15 | 2019-08-27 | 三峡大学 | Chaetomium globosum A causes the application on rotten bacterium medicament as citrus |
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