CN103087923A - Preparation and application of chaetomium globosum and metabolite flavipin thereof - Google Patents
Preparation and application of chaetomium globosum and metabolite flavipin thereof Download PDFInfo
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- CN103087923A CN103087923A CN2012104441644A CN201210444164A CN103087923A CN 103087923 A CN103087923 A CN 103087923A CN 2012104441644 A CN2012104441644 A CN 2012104441644A CN 201210444164 A CN201210444164 A CN 201210444164A CN 103087923 A CN103087923 A CN 103087923A
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Abstract
The invention provides preparation and application of chaetomium globosum and metabolite flavipin thereof. The strain is endophytic fungi obtained by separating gingko leaves, is identified to be chaetomium globosum with a number of CDW7, and is preserved in the China General Microbiological Culture Collection Center with the register number of CGMCC No. 6658. The invention relates to preparation and application of CDW7 metabolite flavipin at the same time. The preparation method comprises the step that the organic solvent extract of the strain CDW7 fermentation broth is subjected to gel column chromatography and recrystallization. The flavipin has strong anti-oxidation activity, and can be used as an antioxidant for application.
Description
Technical field
The present invention relates to preparation and the application of ball hair shell fungi and meta-bolites flavipin thereof.
Background technology
Increasing Free Radical Biology and free radical medical findings show cardiovascular disorder, tumour, senile dementia, aging, and the generation of the diseases such as diabetes, acquired immune deficiency syndrome (AIDS) and development all closely related with active oxygen and free radical, searching and development of new, efficient, safe natural antioxidants are one of important topics of the closely-related major disease research of prevention and treatment and reactive oxygen species and free radicals.
Endophyte of plant refers to those at the certain phase of its life history or all the stage moves in the various tissues of health plant and fungi or the bacterium of organ inside, infected host plant (being temporary transient at least) does not show external illness, can separate from the plant tissue of strict surface sterilization by Histological method or the method that directly amplifies microbial DNA in the plant tissue proves in it and gives birth to.Endogenetic fungus can produce rich and varied secondary metabolite, is considered to the potential resources of active substance, and the oxidation resistant active bacterial strain of screening and active metabolite have caused concern widely from plant endogenesis epiphyte.The Chaetomium secondary fungus metabolite enrich Chemical Diversity, up to now, existing more than 120 kind of secondary metabolite separates from this genus fungal fermentate and obtains, comprise alkaloid, anthraquinone, terpene, the various structures types such as steroidal, wherein there is chemical compound lot all to have significant biological activity, as antitumor, anti-inflammatory, cytotoxicity, enzyme inhibition activity, anti-microbial activity etc.The Chaetomium fungi Chaetomium globosum CDW7 that relates to herein separates from healthy puerarin the endophyte of plant that obtains, its secondary metabolite flavipin (flavipin, 1,2-benzenedicarboxaldehyde-3,4,5-trihydroxy-6-methyl) separated from aspergillus flavipes (Aspergillus flavipes) and scholar's aspergillus (Aspergillus terreus) by Raistrick the earliest, have certain antibiotic and eelworm-killing activity, and its anti-oxidant activity so far there are no the report.
Summary of the invention
One object of the present invention is to provide a plant height to produce the bacterial strain of anti-oxidation active substance flavipin (flavipin).The bacterial strain CDW7 of high yield flavipin provided by the invention (flavipin) is from the endogenetic fungus that separates through the healthy puerarin of surface sterilization, purifying obtains.This bacterial strain is on the PDA plating medium, and the bacterium colony initial stage is white, and gradual change is to darkcyan, back side brown, and the edge is irregular.Scattered or all living creatures of perithecium, avette or subsphaeroidal, the μ m of size (418-610) μ m * (222-277); The ascus shell wall is by the staggered thread organizational composition of dun, and ruff is without barrier film, brown, and meander-like or spirrillum are curling, and there is spinelet on the surface, and the base portion particle size is 3.1 μ m; The thecaspore brown, inferior spherical or lemon shape, the μ m of size (7.5-10.2) μ m * (6.5-7.5) has a leaden hole.Above-mentioned morphological specificity meets Chaetomium (Chaetomium sp.) fungi morphological specificity (seeing accompanying drawing 1, accompanying drawing 2, accompanying drawing 3, accompanying drawing 4).Measure simultaneously its ITS sequence, with HQ914911Chaetomium globosu sequence similarity degree be 100% (seeing accompanying drawing 5), be accredited as chaetomium globosum (Chaetomium globosum, the Genbank accession number is JN588554), strain number CDW7.This bacterial strain is stored in Chinese common micro-organisms culture presevation administrative center (being called for short CGMCC) on October 15th, 2012, and the address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and preserving number is CGMCC No.6658.
Another object of the present invention is preparation method that a kind of flavipin (flavipin) is provided and and application in antioxidant.
Preparation method provided by the invention comprises the steps:
(1) chaetomium globosum (Chaetomium globosum) CDW7 that preserves is inoculated on the PDA flat board, 25 ℃ cultivate 5 days after, get the mycelia piece and put into to sterilize the triangular flask of PDB substratum is housed, in 25 ℃, cultivated 14 days under the 150rpm condition.
(2) with step (1) gained filtering fermentation liquor, filtrate is used dichloromethane extraction, and vacuum concentration obtains fermented product crude extract F1.
(3) the fermented product crude extract F1 in step (2) is carried out Sepahdex LH-20 column chromatography, with chloroform/methanol (volume ratio 1/1) wash-out, each component under wash-out detects through TLC, merge the component that contains flavipin (flavipin), concentrating under reduced pressure gets yellow medicinal extract F2.
Described TLC developping agent is chloroform/methanol (volume ratio 10/1), and the Rf value of flavipin (flavipin) is 0.55
(4) with the recrystallization in acetone/sherwood oil (volume ratio 10/1) of yellow medicinal extract F2 in step (3), namely get compound flavipin (flavipin).
The ability with high yield flavipin (flavipin) that experiment showed, of the present invention, flavipin (flavipin) has very strong anti-oxidant activity, can be used as antioxidant and uses.
Description of drawings
Fig. 1 is that chaetomium globosum (Chaetomium globosum) CDW7 cultivates 10 days colonial morphologies in PDA.
Fig. 2 is that chaetomium globosum (Chaetomium globosum) CDW7 cultivates 25 days colonial morphologies in PDA.
Fig. 3 is chaetomium globosum (Chaetomium globosum) CDW7 perithecium.
Fig. 4 is chaetomium globosum (Chaetomium globosum) CDW7 mycelia and thecaspore.
Fig. 5 is chaetomium globosum (Chaetomium globosum) CDW7 homology phylogenetic tree.
Embodiment
Embodiment one. the separation and purification of endophytic bacterial controlled effect CDW7:
Gather the puerarin of fresh and healthy, be immersed in 30s in 75% alcohol after cleaning, aseptic water washing 3 times, then immerse 3-5%NaClO solution 3-5min is with aseptic water washing 3-5 time.Under aseptic condition, will cut into the fritter of 0.5 * 0.5cm through the puerarin of surface sterilization, be placed in respectively PDA and cultivate based on 25 ℃ of constant temperature culture, after thalline grows, it is consistent that picking colony edge mycelia purifying is cultured to colonial morphology, is stored in the PDA inclined-plane, obtains gingko endogenous bacterium.Wherein a strain strain number is CDW7, now is stored in Chinese common micro-organisms culture presevation administrative center (being called for short CGMCC), and preservation date is on October 15th, 2012, and preserving number is CGMCCNo.6658.
Embodiment two. the evaluation of endophytic bacterial controlled effect CDW7:
Endogenetic fungus CDW7 its on the PDA plating medium, the bacterium colony initial stage is white (seeing accompanying drawing 1), gradual change is to darkcyan (seeing accompanying drawing 2), back side brown, the edge is irregular.Scattered or all living creatures of perithecium, avette or subsphaeroidal, the μ m (seeing accompanying drawing 3) of size (418-610) μ m * (222-277); The ascus shell wall is by the staggered thread organizational composition of dun, and ruff is without barrier film, brown, and meander-like or spirrillum are curling, and there is spinelet on the surface, and the base portion particle size is 3.1 μ m; The thecaspore brown, inferior spherical or lemon shape, the μ m of size (7.5-10.2) μ m * (6.5-7.5) has a leaden hole (seeing accompanying drawing 4).The morphological specificity of above-mentioned CDW7 meets Chaetomium (Chaetomium sp.) fungi morphological specificity.
The mycelia piece of the CDW7 bacterial strain of the dull and stereotyped activation of picking enters in the PDB substratum, cultivates after 5 days for 25 ℃, adopts conventional molecular biology method to extract total DNA.Use round pcr, adopt the general forward primer ITS of fungi
1(5 '-TCCGTAGGTGAACCTGCGG-3 ') and reverse primer ITS
4The ITS of (5 '-TCCTCCGCTTATTGATATGC-3 ') amplification bacterial strain 18S-28S rDNA is interval.After electrophoresis purification and gel recovery, adopt ABI PRISM 3730 sequenators to carry out respectively positive (5 ' → 3 ') and anti-phase (3 ' → 5 ') order-checking.Utilize the ITS rDNA sequence of gained the Blast instrument to carry out the sequence contrast, the sequence similarity degree of finding this sequence and HQ914911Chaetomium globosu is 100%, and being submitted to Genbank database (www.ncbi.nlm.nih.gov/genbank/), the acquisition sequence number is JN588554 (seeing accompanying drawing 5).
Comprehensive morphological is learned and is observed and the molecular biology experiment result, and bacterial strain CDW7 is accredited as chaetomium globosum (Chaetomium globosum).
Embodiment three. the preparation of compound flavipin (flavipin):
(1) fermentation culture of CDW7: the chaetomium globosum CDW7 bacterial classification of getting 4 ℃ of preservations is inoculated on the PDA flat board, cultivated 5 days in 25 ℃ of constant incubators, the cut-off footpath is that 12 pieces of bacterium cake of 0.5cm are put into to sterilize the 250mL triangular flask of 100mL PDB substratum is housed on flat board, cultivates 14 days under 25 ℃, 150rpm condition.
(2) acquisition of fermented product crude extract: get the fermented liquid that above-mentioned steps (1) obtains, with gauze with thalline and separation of fermentative broth, get total ferment filtrate 2L, ferment filtrate is extracted 3-5 time with dichloromethane solvent, combining extraction liquid carries out concentrating under reduced pressure, obtains fermented product crude extract F1 (2g).
(3) separation and purification of compound: the fermented product crude extract that above-mentioned steps (2) obtains is carried out sephadex LH-20 chromatography, with chloroform/methanol (volume ratio 1/1) wash-out, each component under wash-out detects through TLC, merge the component that contains flavipin (flavipin), concentrating under reduced pressure gets yellow medicinal extract F2.Wherein, the TLC developping agent is chloroform/methanol (volume ratio 10/1), and the Rf value of flavipin (flavipin) is 0.55.With yellow medicinal extract F2 recrystallization in acetone/sherwood oil (volume ratio 10/1), namely get compound flavipin (flavipin, 50mg).
Embodiment four. the Structural Identification of compound flavipin (flavipin):
Be yellow crystals with embodiment three compound that obtains, m.p.223-224 ℃, be accredited as flavipin (flavipin according to mass spectrum, nuclear magnetic resonance spectrum, 1,2-benzenedicarboxaldehyde-3,4,5-trihydroxy-6-methyl), spectroscopic data is as follows:
ESI-MS：m/z?197.0560[M+H
+]。
1H-NMR(400MHz，Acetone-d
6)δ(ppm)：2.50(3H，s)，10.38(1H，s)，10.59(1H，s)。
13C-NMR(100MHz，Acetone-d
6)δ(ppm)：10.25(C-7)，113.09(C-2)，124.58(C-6)，129.04(C-1)，136.46(C-4)，150.28(C-3)，151.32(C-5)，192.91(C-9)，197.50(C-8)。
Embodiment five. anti-oxidant experiment: adopt respectively DPPH, FRAP, ABTS and ORAC method to measure flavipin (flavipin) anti-oxidant activity, with BHT and vitamin C as positive control.Test method is as follows
DPPH free radical scavenging method is measured anti-oxidant activity: in 96 orifice plates, add respectively 100 μ L gradient dilution sample solutions and 100 μ L2 * 10
-4DPPH solution (the A of mol/L
Survey), in 100 μ L sample solutions and 100 μ L dehydrated alcohol (A
Sample), 100 μ L DPPH solution and 100 μ L dehydrated alcohol (A
Blank), after 25 ℃ of reaction 30min, use microplate reader to measure 517nm place light absorption value, triplicate is according to formula S C%=[1-(A
Survey-A
Sample)/A
Blank] * 100% calculates clearance rate.Result is with IC
50Expression, namely when the DPPH free radical scavenging activity that reaches 50%, the concentration of analyte sample fluid (antioxidant).
The FRAP method is measured anti-oxidant activity: in 96 orifice plates, add respectively 20 μ L gradient dilution sample solutions and 180 μ LFRAP working fluids (300mmol/L pH 3.6 acetate buffers/10mmol/L TPTZ/20mmol/L FeCl
3, volume ratio 10/1/1, now with the current), measure 593nm place light absorption value, triplicate with microplate reader after 37 ℃ of reaction 4min.With FeSO
47H
2(0.1-1.0mmol/L, n=6, typical curve are Y=1.2273X+0.0460 to the O typical curve, R as a reference
2=0.9994), result is with C
0.5FRAPExpression namely has 0.5mmol/L FeSO
47H
2The sample concentration of O resistance of oxidation.
The ABTS method is measured anti-oxidant activity: ABTS
+By 5mL 7mmol/L ABTS solution and 88 μ L 140mmol/L K
2S
2O
8Aqueous solution lucifuge reaction generated after 12 hours, with alcohol dilution (732nm place light absorption value be 0.70 ± 0.02).Get 20 μ L gradient dilution sample solutions and be added to 180 μ LABTS
+Measure the 732nm light absorption value after lucifuge reaction 10min in solution, net result is with IC
50Expression namely ought reach 50% ABTS
+During free radical scavenging activity, the concentration of analyte sample fluid (antioxidant).
The ORAC method is measured anti-oxidant activity: configuration 160mmol/L AAPH phosphate buffer solution (pH=6.9) and 4 * 10
-3Mmol/L uranin phosphate buffer solution (pH=6.9), the lucifuge cryopreservation dilutes 10000 times with phosphate buffer solution before using.Add the uranine liquid of 25 μ L gradient dilution sample liquid and 150 μ L in 96 hole black microwell plates, add again 25 μ L AAPH to bring out fluorescent quenching, (parameters is: excitation wavelength 485nm, emission wavelength 528nm, 37 ℃ to use SpectraMax M5 fluorescence microplate reader; Cycle number: 35; Loop cycle: 120s) measure.Experimental result represents with ORAC (ORAC).The ORAC index refers under antioxidant exists; the size of protected integral area on the fluorescence decline curve (needing the protected area of the unified 1 μ mol/L Trolox of conversion), 1 ORAC unit definition is: final concentration is the protection integral area of Trolox correspondence on the fluorescence decline curve of 1 μ mol/L.
The anti-oxidant test result of compound flavipin (flavipin) is as shown in table 1:
Table 1 compound flavipin (flavipin) anti-oxidant activity
The letter subscript represents 5% horizontal significant difference
Above-mentionedly experiment showed, that compound flavipin (flavipin) has the anti-oxidant activity suitable with vitamin C, it has huge potentiality as novel antioxidant.
Claims (3)
1. gingko endogenous fungus chaetomium globosum (Chaetomium globosum) CDW7 of flavipin (flavipin) is produced in a strain, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, accession designation number is CGMCC No.6658.
2. method for preparing flavipin (flavipin) is characterized in that this compound adopts following steps to obtain:
(1) the chaetomium globosum CDW7 that preserves is inoculated on the PDA flat board, cultivates after 5 days for 25 ℃, get the mycelia piece in the PDB substratum, 25 ℃, cultivated 14 days under the 150rpm condition.
(2) with step (1) gained filtering fermentation liquor, filtrate is used dichloromethane extraction, and concentrating under reduced pressure obtains fermented product crude extract F1.
(3) the fermented product crude extract F1 in step (2) is carried out Sepahdex LH-20 column chromatography, with chloroform/methanol (volume ratio 1/1) wash-out, detect through TLC, merge the component that contains flavipin (flavipin), concentrating under reduced pressure gets yellow medicinal extract F2.
(4) with the recrystallization in acetone/sherwood oil (volume ratio 10/1) of yellow medicinal extract F2 in step (3), namely get compound flavipin (flavipin).
3. the application of a flavipin claimed in claim 2 (flavipin) in the preparation antioxidant.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105838613A (en) * | 2015-01-14 | 2016-08-10 | 东北林业大学 | Pigeon pea endophytic fungi high in yield of flavipin and application of pigeon pea endophytic fungi |
| CN105861325A (en) * | 2016-04-28 | 2016-08-17 | 三峡大学 | Chaetomium globosum, antioxidant and bacteriostatic agent prepared from chaetomium globosum and application |
| CN110129376A (en) * | 2019-03-08 | 2019-08-16 | 浙江工业大学 | Gingko endogenous fungus metabolite and preparing the application in antioxidant |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102742605A (en) * | 2012-06-06 | 2012-10-24 | 吉林大学 | Application of gingko endogeny Chaetomium globosum in prevention and control of plant pathogenic fungi |
| CN102742606A (en) * | 2012-06-06 | 2012-10-24 | 吉林大学 | Application of gingko endogeny Chaetomium globosum in prevention and control of plant pathogenic fungi |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102742605A (en) * | 2012-06-06 | 2012-10-24 | 吉林大学 | Application of gingko endogeny Chaetomium globosum in prevention and control of plant pathogenic fungi |
| CN102742606A (en) * | 2012-06-06 | 2012-10-24 | 吉林大学 | Application of gingko endogeny Chaetomium globosum in prevention and control of plant pathogenic fungi |
Non-Patent Citations (4)
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| 《Current Opinion in Biotechnology》 20050430 Sameer AS Mapari "Exploring fungal biodiversity for the production of water-soluble pigments as potential natural food colorants" 第16卷, 第2期 * |
| 《安徽农业科学》 20091231 谢辉 "1株杜仲内生真菌的抗氧化活性分析和菌种鉴定" 第37卷, 第1期 * |
| SAMEER AS MAPARI: ""Exploring fungal biodiversity for the production of water-soluble pigments as potential natural food colorants"", 《CURRENT OPINION IN BIOTECHNOLOGY》, vol. 16, no. 2, 30 April 2005 (2005-04-30) * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105838613A (en) * | 2015-01-14 | 2016-08-10 | 东北林业大学 | Pigeon pea endophytic fungi high in yield of flavipin and application of pigeon pea endophytic fungi |
| CN105861325A (en) * | 2016-04-28 | 2016-08-17 | 三峡大学 | Chaetomium globosum, antioxidant and bacteriostatic agent prepared from chaetomium globosum and application |
| CN110129376A (en) * | 2019-03-08 | 2019-08-16 | 浙江工业大学 | Gingko endogenous fungus metabolite and preparing the application in antioxidant |
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