CN105779298B - One plant of E.amstelodami and benzofuran compounds and preparation method thereof - Google Patents
One plant of E.amstelodami and benzofuran compounds and preparation method thereof Download PDFInfo
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- CN105779298B CN105779298B CN201410818028.6A CN201410818028A CN105779298B CN 105779298 B CN105779298 B CN 105779298B CN 201410818028 A CN201410818028 A CN 201410818028A CN 105779298 B CN105779298 B CN 105779298B
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Abstract
The invention discloses one plant of E.amstelodami, the deposit number of the E.amstelodami is CCTCC NO:M 2014413.The invention also discloses a kind of methods for preparing benzofuran compounds, this method includes that the E.amstelodami that deposit number is CCTCC NO:M 2014413 ferments, obtain the fermentation culture medium containing benzofuran compounds, wherein, shown in the structural formula of the benzofuran compounds such as following formula (I).In addition, the invention also discloses structural formula benzofuran compounds as shown in formula (I).E.amstelodami of the invention, which can ferment, obtains benzofuran compounds, and the benzofuran compounds of acquisition are practically insoluble in water, may be used as yellow pigment.
Description
Technical field
The present invention relates to one plant of E.amstelodami (Eurotium amstelodami) and benzofurans chemical combination
Object and preparation method thereof, and in particular, to one plant of E.amstelodami prepares benzo using the E.amstelodami
The method and benzofuran compounds of furfuran compound.
Background technique
Bulk bacteria are distributed widely in some traditional fermented foods of states such as natural environment, and China, South Korea and Japan
Important leavening, such as they are the dominant bacterias of the mouldy technique of Japanese stripped tuna and Chinese Fu-brick tea fungus growing process.As it is a kind of with
The closely related Common fungi of human lives, people have been devoted to understand distribution feelings of the bulk bacteria in various ecological environments
(known mainly include anthraquinone-derivative species pigment, benzaldehyde derivative to the secondary metabolite of condition, discovery and research bulk bacteria
Class pigment and diketopiperazines mycotoxin etc.), to illustrate bulk bacteria in the function value or safety of field of food industry
Property etc., this is also the important content of natural products the Study on Resources exploitation.
Summary of the invention
The object of the present invention is to provide one plant of new E.amstelodami and benzofuran compounds and its preparations
Method.
To achieve the goals above, on the one hand, the present invention provides one plant of E.amstelodami, the Amsterdam
The deposit number of bulk bacteria is CCTCC NO:M 2014413.
On the other hand, the present invention provides a kind of method for preparing benzofuran compounds, this method includes by preservation
Number is that the E.amstelodami of CCTCC NO:M 2014413 ferments, and is obtained containing benzofuran compounds
Fermentation culture medium, wherein shown in the structural formula of the benzofuran compounds such as following formula (I):
In another aspect, the present invention provides structural formula benzofuran compounds as shown in formula (I).
Through the above technical solutions, E.amstelodami of the invention, which can ferment, obtains benzofurans chemical combination
The benzofuran compounds of object, acquisition are practically insoluble in water, may be used as yellow pigment.
Other features and advantages of the present invention will the following detailed description will be given in the detailed implementation section.
Biological deposits
E.amstelodami (Eurotium amstelodami) of the invention, was preserved on September 14th, 2014
In China typical culture collection center (address: wuchang, wuhan Luo Jia Shan, Wuhan University, postcode: 430072) (preservation
Unit is abbreviated as CCTCC), deposit number is CCTCC NO:M 2014413.
Detailed description of the invention
The drawings are intended to provide a further understanding of the invention, and constitutes part of specification, with following tool
Body embodiment is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 is the form photo of E.amstelodami of the invention, wherein Fig. 1-A to Fig. 1-C is microscope photograph
Piece, Fig. 1-D indicate the bacterium colony photo of plate culture；
Fig. 2 is benzofuran compounds of the invention1H-13C correlation map (HMBC) and1H-1H correlation map
(COSY), wherein filament indicates HMBC spectrum, and thick line indicates COSY spectrum；
Fig. 3 is the spectral scan curve graph of the benzofuran compounds of the invention measured by ultraviolet specrophotometer.
Specific embodiment
Detailed description of the preferred embodiments below.It should be understood that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
In the present invention, in the absence of explanation to the contrary, the various experiments in the present invention carry out under normal pressure；It relates to
And each substance (especially column chromatographs the silica gel and gel that use) weight in terms of butt.
The present invention provides one plant of E.amstelodami, the deposit number of the E.amstelodami is CCTCC
NO:M 2014413.
The conidium of E.amstelodami (laboratory is named as TIB.BPE.12001) of the invention is in grayish green
Color, bacterium colony reverse side are in yellowish-brown, and ascus is subsphaeroidal, ascospore biconvex mirror shape, have ditch, rough surface, form among two valves
(in Fig. 1, Fig. 1-A, Fig. 1-B and Fig. 1-C are that E.amstelodami is cultivated in solid PDA medium to feature as shown in Figure 1
Micro- sem observation photo after 4 days, Fig. 1-D are that PDA plate bacterium colony observes photo).
It is CCTCC NO:M that the method provided by the invention for preparing benzofuran compounds, which includes by deposit number,
2014413 E.amstelodami ferments, and obtains the fermentation culture medium containing benzofuran compounds, wherein
Shown in the structural formula of the benzofuran compounds such as following formula (I):
According to the present invention, the mode of the fermentation is not particularly limited, it is preferable that the mode of the fermentation is in 20-
Stationary culture 20-40 days at 25 DEG C.
According to the present invention, the fermentation is preferably solid fermentation, and therefore, the culture medium that the fermentation uses contains 1 weight
The substrate of part and the water of 1-1.5 parts by weight are (that is, the culture medium that fermentation uses contains substrate and water, and the weight ratio of substrate and water
For 1:1-1.5), the substrate is preferably in wheat, barley, naked oats, oat, polished rice, long-grained nonglutinous rice, corn, glutinous rice, Gorgon fruit and Semen Coicis
At least one.Relative to the culture medium that every gram of fermentation uses, the inoculum concentration of the E.amstelodami is 104-108
It is a.
In the present invention, the method also includes successively carrying out activation and seed training to E.amstelodami before fermentation
It supports, activation is so that it is restored fermenting property in order to which the strain of preservation state is put into suitable culture medium and is cultivated；Seed culture
It is pure and strong culture more in order to obtain, that is, is flushed, is inoculated with the enough E.amstelodamis of quantity.
It can adopt and carry out activation and seed culture with the conventional methods in the field, for example, the activation may include by Amsterdam
The mycelium of bulk bacteria is seeded on PDA plate, is cultivated 3-5 days at 28-32 DEG C.The seed culture may include after activating
E.amstelodami be seeded in seed culture medium (liquid), 28-32 DEG C culture 2-3 days.PDA plate and seed culture
Base is well known to the skilled person, and details are not described herein.
According to the present invention, the method can also include that benzofuran compounds are purified from fermentation culture medium, described
Purifying the following steps are included:
(1) fermentation culture medium is extracted with organic solvent, the organic solvent is methanol, ethyl alcohol, n-butanol, positive fourth
At least one of ketone, acetone, methylene chloride, chloroform, ethyl acetate and methyl acetate；
(2) product extracted is subjected to column chromatography for separation.
Preferably, in step (1), the temperature of the extraction is 20-35 DEG C, time 10-30h.
Preferably, in step (2), the column chromatography for separation includes alternately silica gel column chromatography and gel filtration chromatography.
It is highly preferred that the mode for carrying out column chromatography is that alternately silica gel column chromatography and gel filtration chromatography be twice.For the first time
Silica gel column chromatography collects the fraction for the elution for being 100:0-99:1 by methylene chloride and methanol volume ratio, second of silica gel
Column chromatography collects the fraction for the elution for being 1.5-2.5:1 by petroleum ether and chloroform volume ratio, and gel filtration chromatography is equal twice
Collect the fraction eluted by methanol.
In the present invention, the weight ratio of silica gel used in silica gel column chromatography and benzofuran compounds to be purified is preferred
For 20-50:1.The granular size of the silica gel used is preferably 200-300 mesh.In silica gel column chromatography, benzofurans to be purified
The time that compound is contacted with silica gel is preferably 0.5-3h.
In the present invention, the weight ratio of gel and benzofuran compounds to be purified that gel filtration chromatography uses is preferably
60-150:1.The carrying capacity of the gel used is preferably 250-350mg/mg gel.The gel used can be sephadex
Glue (such as Sephadex G or Sephadex LH-20), preferably hydroxypropyl sephadex (such as Sephadex LH-20).It is solidifying
In plastic column chromatography, the time of benzofuran compounds to be purified and gel contacts is preferably 0.5-3h.
When carrying out gel filtration chromatography, automatic collector can be used and collect eluent, and examined using thin-layer chromatography
It surveys, the stationary phase that thin-layer chromatography uses can be silica gel, aluminium oxide or cellulose, and mobile phase can be the stone that volume ratio is 3:1
The methylene chloride-methanol of oily ether-ethyl acetate, 10:1 petroleum ether-acetone or 20:1, thin-layer chromatography show yellow band, then receive
Collect and merge the eluent of the part.When carrying out gel filtration chromatography, the mistake that retention volume is 70-80mL can also be directly collected
Eluent after column, and it is used for subsequent step.
According to the present invention, it will be appreciated to those of skill in the art that during column chromatography for separation, what back obtained is washed
Before de- liquid is for the column chromatography of next step, need that according to circumstances suitably eluent is concentrated, and herein no longer to this
It is repeated.
The present invention also provides a kind of benzofuran compounds, the structural formula of the benzofuran compounds such as above formulas
(I) shown in.
The molecular formula of benzofuran compounds provided by the invention is C19H22O3, it is not soluble in water, it is in yellow powder, it can
For use as yellow pigment.
The present invention will be described in detail by way of examples below.
In following embodiment, 200-300 mesh silica gel is purchased from Qingdao Marine Chemical Co., Ltd.；Gel is purchased from Merck company.
Embodiment 1
The present embodiment is used to illustrate the preparation method of benzofuran compounds of the present invention.
(1) fermented and cultured
(1) bacterial strain activates: by E.amstelodami TIB.BPE.12001 (CCTCC NO:M 2014413) mycelium
It is inoculated on PDA plate, 30 DEG C are cultivated 4 days.PDA plate: by potato 200g, glucose 20g, agar 15g adds distilled water extremely
1L, 121 DEG C of high pressure steam sterilization 20min, is made plate.
(2) seed culture: after bacterium covers with PDA plate, by 5 1cm2The lawn of size is together with culture medium inoculated to containing
In the 500mL triangular flask of 200mL seed culture medium, on 120rpm shaking table, 30 DEG C are cultivated 3 days, obtain seed culture fluid.It is described
Every liter of seed culture medium contains: glucose 30g, the KH of yeast extract 20g, 2g2PO4, the MgSO of 2g4·7H2O, surplus are water, pH
Value is 6.0；121 DEG C sterilize 20 minutes.
(3) ferment: by seed culture fluid by volume 1:20 ratio access solid medium in, in 25 DEG C of stationary cultures
30 days, obtain solid fermentation culture；The solid medium is made of 320g rice and 480mL distilled water；By 320g rice
It is added in 2L triangular flask (totally 20 bottles) with 480mL pure water, soaked overnight, 121 DEG C of high pressure steam sterilization 30min, it is cooling stand-by.
(2) compound isolates and purifies
(1) solid fermentation culture is smashed to pieces with instrument, ethyl acetate is added and extracts, is standing and soaking 1 day, obtains
To extracting solution, the extracting solution is evaporated under reduced pressure, obtains crude extract 10.8g.
(2) crude extract is through silica gel column chromatography (200-300 mesh silica gel, 6 × 40cm of Ф), with methylene chloride-methanol volume ratio
It is eluted for 100:0-99:1, coutroi velocity 5mL/min collects the fraction A of elution.
(3) after fraction A decompression spin concentration (3mL) carry out gel Sephadex LH-20 column chromatography (Ф 1 ×
100cm), it is eluted with methanol, coutroi velocity 0.5mL/min, collects eluent, thin-layer chromatography inspection with automatic collector
It surveys, merges the component with yellow band, obtain A3.A3 is through silica gel column chromatography (200-300 mesh silica gel, 1.5 × 30cm of Ф) body
Product is concentrated into 3mL again through gel Sephadex LH- after eluting (coutroi velocity 5mL/min) than the petroleum ether-chloroform for being 2:1
20 columns chromatograph (1 × 100cm of Ф), and coutroi velocity 0.5mL/min obtains meoh eluate, then concentrated are dried to obtain yellow
Powder (compound 1) 17.3mg.
(3) characterization of compound
The product (compound 1) of step (2) is subjected to infrared spectroscopy (instrument model is Bruker Tensor 27), is had
(instrument model is 600MHz Bruker for machine mass spectrum (instrument model is Bruker microTOF-QII) and nuclear magnetic resoance spectrum
Avance III) analysis.The characterize data of product is as follows:
Yellow powder；
Organic mass spectrometry obtains high resolution mass spectrum HRESIMS:m/z 321.1572 [M+Na]+(calcd.for
C19H22O3Na, 321.1569)；
Nmr analysis obtain hydrogen spectrum (1H-NMR) and carbon spectrum (13C-NMR) it is shown in Table 1；
The HMBC spectrum and COSY spectrum that nmr analysis obtains are shown in Fig. 2.
1 hydrogen of table spectrum (1H-NMR) and carbon spectrum (13C-NMR) data
| Number | δH | δC |
| 1 | 110.8(s) | |
| 2 | 128.2(s) | |
| 3 | 148.6(s) | |
| 4 | 7.47(s) | 119.5(d) |
| 5 | 125.4(s) | |
| 6 | 157.3(s) |
| 7 | 10.23(s) | 193.1(d) |
| 1' | 6.68(s) | 98.5(d) |
| 2' | 162.7(s) | |
| 3' | 2.75(t,7.5) | 28.4(t) |
| 4' | 2.21(q,6.8) | 32.3(t) |
| 5' | 5.43(dt,14.3,6.5) | 130.0(d) |
| 6' | 5.52(m) | 126.9(d) |
| 7' | 1.68(d,7.2) | 17.8(q) |
| 1″ | 3.42(d,7.4) | 27.6(t) |
| 2″ | 5.31(t,7.4) | 121.2(d) |
| 3″ | 133.8(s) | |
| 4″ | 1.79(s) | 25.6(q) |
| 5″ | 1.71(s) | 17.8(q) |
| 6-OH | 11.62(s) |
Note: (CDCl3) δ: ppm, J:Hz, solvent C DCl3,1H:600MHz,13C:150MHz。
Comprehensive high resolution mass spectrum, hydrogen spectrum, carbon spectrum, HMBC spectrum and COSY spectrum can release structure such as formula (I) institute of compound 1
Show, molecular formula are as follows: C19H22O3, molecular weight are as follows: 298.
(4) performance test of compound
Compound 1 is configured to the test solution that concentration is 0.15mg/mL with methanol, through TU-1810 type ultraviolet specrophotometer
Measurement, obtain compound 1 has strong absworption peak at wavelength 230nm, 275nm, 385nm (referring to Fig. 3)；Moreover, the compound is in
Yellow；Solubility under conditions of 25 DEG C and 1atm in water is less than 0.01g/100cm3, it is insoluble in water；Illustrate that it can be used as
Yellow pigment.
As can be seen from the above embodiments, E.amstelodami of the invention can ferment shown in acquisition formula (I)
Benzofuran compounds, the compound can be used as yellow pigment.
Moreover, the light resistance and resistance to oxidation of benzofuran compounds of the invention are also by further experiment discovery
Originality is relatively strong.
It is described the prefered embodiments of the present invention in detail above in conjunction with attached drawing, still, the present invention is not limited to above-mentioned realities
The detail in mode is applied, within the scope of the technical concept of the present invention, a variety of letters can be carried out to technical solution of the present invention
Monotropic type, these simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can
No further explanation will be given for the combination of energy.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (14)
1. one plant of E.amstelodami (Eurotium amstelodami), the deposit number of the E.amstelodami
For CCTCC NO:M 2014413.
2. a kind of method for preparing benzofuran compounds, which is characterized in that it is CCTCC that this method, which includes by deposit number,
The E.amstelodami of NO:M 2014413 ferments, and obtains the fermentation culture medium containing benzofuran compounds,
Wherein, shown in the structural formula of the benzofuran compounds such as following formula (I):
3. according to the method described in claim 2, wherein, the mode of the fermentation is the stationary culture 20-40 at 20-25 DEG C
It.
4. according to the method in claim 2 or 3, wherein it is described fermentation use culture medium contain 1 parts by weight substrate and
The water of 1-1.5 parts by weight, the substrate are wheat, barley, naked oats, oat, polished rice, long-grained nonglutinous rice, corn, glutinous rice, Gorgon fruit and Semen Coicis
At least one of.
5. according to the method described in claim 2, wherein, the method also includes purifying benzofurans from fermentation culture medium
Compound, it is described purifying the following steps are included:
(1) fermentation culture medium is extracted with organic solvent, the organic solvent be methanol, ethyl alcohol, n-butanol, positive butanone,
At least one of acetone, methylene chloride, chloroform, ethyl acetate and methyl acetate；
(2) product extracted is subjected to column chromatography for separation.
6. according to the method described in claim 5, wherein, the column chromatography for separation includes alternately silica gel column chromatography and gel
Column chromatography.
7. according to the method described in claim 6, wherein, carrying out the mode of column chromatography as alternately silica gel column chromatography and gel
Column chromatographs twice, the elution that it is 100:0-99:1 by methylene chloride and methanol volume ratio that first time silica gel column chromatography, which is collected,
Fraction, second silica gel column chromatography collect the fraction for the elution for being 1.5-2.5:1 by petroleum ether and chloroform volume ratio,
Gel filtration chromatography collects the fraction eluted by methanol twice.
8. method according to claim 6 or 7, wherein silica gel used in silica gel column chromatography and benzo furan to be purified
The weight ratio of class of muttering compound is 20-50:1.
9. according to the method described in claim 8, wherein, the granular size of the silica gel used is 200-300 mesh.
10. method according to claim 6 or 7, wherein in silica gel column chromatography, benzofuran compounds to be purified
The time contacted with silica gel is 0.5-3h.
11. method according to claim 6 or 7, wherein the gel and benzofuran to be purified that gel filtration chromatography uses
The weight ratio of class compound is 60-150:1.
12. according to the method for claim 11, wherein the carrying capacity of the gel used is 250-350mg/mg gel.
13. according to the method for claim 11, wherein the gel used is hydroxypropyl sephadex.
14. method according to claim 6 or 7, wherein in gel filtration chromatography, benzofuran compounds to be purified
Time with gel contacts is 0.5-3h.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103880826A (en) * | 2013-12-10 | 2014-06-25 | 中山大学 | Isobenzofuranone compounds as well as preparation method and application thereof |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103880826A (en) * | 2013-12-10 | 2014-06-25 | 中山大学 | Isobenzofuranone compounds as well as preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| filamentous fungi are large-scale producers of pigments and colorants for the food industry;laurent dufosse et al,;《Sciencedirect》;20140430;第56-61页 * |
| 散囊菌黄色素的提取及稳定性研究;王波等;《生物学杂志》;20090630;第63-65页 * |
| 茯砖茶中冠突散囊菌的次级代谢产物及其生物活性研究;彭晓赟等;《中草药》;20130731;第44卷(第14期);摘要，第1883页右栏第2段 * |
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