CN104450818B - A kind of method and its application that pyrroles's acid compound is prepared using hickory chick - Google Patents
A kind of method and its application that pyrroles's acid compound is prepared using hickory chick Download PDFInfo
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Abstract
The invention belongs to natural drug separation and Extraction field, and in particular to a kind of method and its application that pyrroles's acid compound is prepared using hickory chick.Specifically preparation method is：Using hickory chick as raw material, through solid fermentation, the organic extraction of product, isolate and purify to obtain pyrroles's acid compound.Hickory chick raw material used is hickory chick M.co 0010（Morehella esculenta）, Wuhan, China Wuhan University China typical culture collection center is preserved on July 6th, 2014, deposit number is CCTCC M 2014324.A kind of pyrroles's acid compound is obtained according to the preparation method, experiment proves that pyrroles's acid compound has antioxidation activity, can be used for preparing anti-oxidation medicine.Pyrroles's acid compound prepared by the present invention has preferable antioxidation activity, can utilize bacterial strainMorehella esculentaFermentation is made, and culture medium raw material derives from a wealth of sources, and preparation method is simple, industrialized production easy to implement.
Description
Technical field
The invention belongs to natural drug separation and Extraction field, and in particular to one kind prepares pyrroles's acid compound using hickory chick
Method and its application.
Background technology
Natural Antioxidants, which have, removes free radical, and then has the function that anti-aging and prevent various diseases.Eat medicine
The nutriments such as amino acid, vitamin, polysaccharide, protein, and its secondary metabolite structure are not only rich in macro fungi
Type is various, these cometabolisms have anti-oxidant, antibacterium, promote nerve growth factor synthesis, enzyme inhibitor, it is antimycotic,
The multiple biological activities such as cell toxicant, receptor antagonist.Anti-oxidation function is one of critical function of edible and medical fungi, from edible and medical fungi
Middle separation and Extraction Natural Antioxidants, contribute to the utilization of edible and medical fungi resource, enrich natural pharmaceutical resources, promote
Human health develops and disease prevention.Based on background above, carry out the research of the present invention.
The content of the invention
It is an object of the invention to for containing abundant Natural Antioxidants, but development and utilization level in edible mushroom
It is low；A kind of method and its application that pyrroles's acid compound is prepared using hickory chick are provided.Pyrroles's acid compound prepared by the present invention
There is preferable antioxidation activity, and culture medium raw material derives from a wealth of sources, preparation method is simple, industrialized production easy to implement.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of hickory chick, the strain are hickory chick M.co 0010（Morehella esculenta）, in 2014 7
The moon is preserved in Wuhan, China Wuhan University China typical culture collection center on the 6th, and deposit number is CCTCC M
2014324。
A kind of method that pyrroles's acid compound is prepared using hickory chick, using hickory chick as raw material, is had through solid fermentation, product
Machine extraction, isolate and purify to obtain pyrroles's acid compound.
The method that pyrroles's acid compound is prepared using hickory chick, is comprised the following steps：
（1）Solid fermentation：Solid fermentation is carried out after hickory chick is activated；Then cultivated；
（2）The organic extraction of product：By step（1）Obtained mycelium is cultivated to be extracted with organic solvent with culture medium；Extraction
After liquid concentration, extracted with isometric ethyl acetate and water；Ethyl acetate phase is taken, after being dehydrated, concentrating, obtains organic coarse
Extract medicinal extract；
（3）Isolate and purify：After organic crude extract medicinal extract is dissolved with methanol, gel filtration chromatography is carried out；Using thin-layer chromatography-
Bioautography（Bibliography：Three kinds of Chinese medicines such as the application Thin Layer Chromatography-Autoradiography such as Gu Lihua technology evaluation root of three-nerved spicebush
Antioxidation activity Acta Pharmaceutica Sinicas 2006,41 (10):956-962）The antioxidation activity of each component is detected, there will be antioxygen
After changing active Fraction collection, merging, then reversed-phase silica gel column chromatography is carried out, collect the component with antioxidation activity；Again finally
Using normal-phase silica gel column chromatography, pyrroles's acid compound with antioxidation activity is obtained.
Step（1）The culture medium prescription composition of described its condition solid fermentation of culture is：Potato 16-25wt%, grape
Sugared 1.5-2.5wt%, peptone 0.5-1.5wt%, 1-2wt% agar, surplus is water；Natural pH, 0.1 Mpa, 100 ~ 140 DEG C go out
15 ~ 40 min of bacterium, in 25-28 DEG C of constant temperature incubation 25-35 days.
Step（2）The organic solvent is ethyl acetate, the one or more in methanol, acetone, glacial acetic acid.
Step（3）Its parameter of the gel filtration chromatography is：Sephadex LH-20 sephadex columns, methanol elution,
Flow velocity is 12 s/ drops, and often pipe collects 5-10mL, and per test tube, sampling carries out thin-layer chromatography：Solvent is chloroform:Methanol=10:1,
Color developing agent is the sulfuric acid ethanol of 10wt%, and similar compositions merge.
Step（3）Its elution of the reversed-phase silica gel column chromatography is：Eluant, eluent, methanol are used as by the use of the mixed liquor of methanol and water
The 5-30% of mixeding liquid volume is accounted for, contains the formic acid that volume fraction is 0.1-1%, progress gradient elution in mixed liquor；It is described just
Its elution of phase silica gel column chromatography is：Eluant, eluent, volume ratio 40 are used as by the use of the mixed liquor of chloroform and methanol:1-120:1, eluant, eluent
In contain volume fraction be 0.1-2 ‰ formic acid.
Pyrroles's acid compound that a kind of method as described above obtains, its molecular formula is C5H5NO2, structural formula is：
。
The application of pyrroles's acid compound, is specially：It is used to prepare anti-oxidation medicine.
The beneficial effects of the present invention are：
Pyrroles's acid compound prepared by the present invention has preferable antioxidation activity, can utilize bacterial strainMorehella esculentaFermentation is made, and culture medium raw material derives from a wealth of sources, and preparation method is simple, industrialized production easy to implement.
Brief description of the drawings
The thin-layer chromatogram of Fig. 1 compound pyrroles's -2- carboxylic acids；1 represents control vitamin C, and 2 represent compound pyrroles -2-
Carboxylic acid.
Embodiment
The present invention further illustrates the present invention with the following example, but protection scope of the present invention is not limited to following reality
Apply example.
The preparation method of pyrroles's acid compound provided by the invention, its step are：
1) solid fermentation and the organic extraction of product：By hickory chick（Morehella esculenta）Slant strains are first being trained
Support and activated on tablet, 26 DEG C of cultivation temperature, incubation time 10 days, then large batch of solid fermentation is carried out, using culture medium prescription
For：200 g containing potato in every liter of water, 20 g of glucose, 10 g of peptone, agar 10 g, pH naturally, 0.1 Mpa, 100 ~
140 DEG C of 15 ~ 40 min of sterilizing, are placed in 25-28 DEG C of constant temperature incubation.After fermentation 25-35 days, with centrifugal process by mycelium and zymotic fluid
Separation.Mycelium is extracted with culture medium ethyl acetate, methanol, acetone and other organic solvent after culture.Second is used after leaching liquor concentration
Acetoacetic ester and water equal-volume extraction, concentrate after ethyl acetate phase dehydration, obtain organic crude extract medicinal extract.
2) separating-purifying：1) organic crude extract medicinal extract described in is dissolved with methanol, Sephadex LH-20 glucans coagulate
Plastic column chromatography, methanol elution, flow velocity is about 12 s/ drops, and often pipe collects 5-10 mL.Using thin-layer chromatography-bioautography inspection
The antioxidation activity of every pipe is surveyed, merges active component, is dissolved with methanol, carries out reversed-phase silica gel column chromatography, with containing body
Fraction for 0.1-1% formic acid methanol and water mixed liquor as eluant, eluent, methanol accounts for the 5-30% of mixeding liquid volume, progress
Gradient elution, is in charge of collection, merges the component of antioxidation activity；Finally chromatographed using purification on normal-phase silica gel, be with containing volume fraction
The chloroform of ‰ formic acid of 0.1-2 and the mixed liquor of methanol are as eluant, eluent, volume ratio 40:1-120:1, obtain that there is anti-oxidant work
The pyrrole acid pure compounds of property.
X-ray single crystal diffraction and nuclear magnetic resonance spectroscopy are carried out to sterling, and carry out antioxidation activity test.
According to X-ray single crystal diffraction and nuclear magnetic resonance data, Structural Identification is carried out to compound, it may be determined that the knot of compound
Structure.
Compound of the present invention is pyrroles's acid compound pyrroles's -2- carboxylic acids, molecular formula C5H5NO2, structural formula is such as
Under：
。
According to the Antioxidative Activity Determination of compound pyrroles's -2- carboxylic acids, it is found that it is stronger anti-oxidant pyrroles -2- carboxylic acids have
Activity（Embodiment 3）.
Embodiment 1
With potato glucose solid medium（200 g containing potato in every liter of water, 20 g of glucose, 10 g of peptone,
Agar 10 g, pH are naturally, 0.1 MPa, 121 DEG C of 30 min of sterilizing）, by the bacterial strain after activationMorehella esculentaGu
9.6 L of body culture, in 26 DEG C of 30 d of shaking table culture.Mycelium and culture medium ethyl acetate after culture：Methanol：Glacial acetic acid body
Product is than being 80：15：5 organic solvent extraction.Taken off after leaching liquor concentration with ethyl acetate and water equal-volume extraction, ethyl acetate phase
Concentrated after water, obtain methanol soluble crude extract (1.54 g, brown medicinal extract).
By 1.54 g methanol crude extracts in previous step, fully dissolved with proper amount of methanol, carry out Sephadex LH-20 Portugals
Polysaccharide gel column chromatography, methanol elution, flow velocity is about 12 s/ drops, and often pipe collects 5mL, and per test tube, sampling carries out thin-layer chromatography（Exhibition
It is chloroform to open agent：Methanol=10：1, color developing agent：10% sulfuric acid ethanol）Analysis, similar compositions merge, and obtain A (29.5 mg), B
(115.7 mg), C (38.9 mg), D (26.1 mg), E (1013.9 mg) and F (240.4 mg) component.Using thin-layer chromatography-
Antioxidation activity of the bioautography detection per component, obtains the component F (240.4 mg) with antioxidation activity.To component
F carries out reverse phase silica gel column (30 g) chromatography, and F1.1 is afforded with 400 ml, 30% methanol-waters（16.7 mg）And F1.2（11.7
mg）.Component F1.1 recycles Sephadex LH-20 sephadex column layers, is eluted with the acetone containing 1 ‰ formic acid, and flow velocity is about
12 s/ drops, 2.5 mL of often pipe collection, obtain F1.1.1（8 mg）.Component F1.1.1 finally chromatographs 80 ml chlorine using purification on normal-phase silica gel
Imitation-carbinol (80：1, contain 1 ‰ formic acid in chloroform) afford the pure compounds (1.5 with antioxidation activity
mg)。
The method that the structure determination of compound can use X-ray single crystal diffraction.Spread out according to the X-ray monocrystalline of the compound
Penetrate data, it may be determined that the structure of the compound.
The X-ray single crystal diffraction data of the compound：Monocline day crystallographic system, crystal size：0.7 × 0.7 × 0.4 mm, it is empty
Between group be C 2/c, cell parameter：a=13.2599 (14) ×10-10M,b= 5.0253(5) ×10-10M,c =
14.8921(16) ×10-10M, α=90 °, β=99.199 (11) °, γ=90 °,V =979.57(18) ×10-30 m3,Z =
4,Dc = 1.507 g∙cm-3,μ(MoKα) = 0.119㎜-1, solved by direct method, with least square method refine, the chemical combination
The structure of thing is determined.
Embodiment 2
Similar to Example 1, difference lies in Identification of chemical structure to use NMR spectrum for it（1H-NMR、13C-NMR、
HSQC、HMBC、1H,1H-COSY）Analytic method, the structure of compound is can determine that according to NMR data.
The compound1H NMR [500 MHz, (CD3)2CO]：δ 6.85 (m, 1H, H-3), δ 6.21 (m,
1H, H-4), δ 7.05 (m, 1H, H-5).The compound13C NMR [500 MHz, (CD3)2OD]：δ 124.0
(s,C-2), δ 115.8 (d, C-3), δ 110.4 (d,C-4), δ 124.1 (d, C-5), δ 162.0 (s, C-
6).From compound1H-NMR and13The chemical displacement value of C-NMR shows to contain mono-substituted pyrrole structure unit in compound.
According to another13162.0 s of C-NMR chemical displacement values δ speculate that it is the carboxyl for being connected in No. 2 positions of pyrrole ring.
Embodiment 3
Using thin-layer chromatography-bioautography（Bibliography：Gu Lihua, waits applications thin-layer chromatography-biology certainly aobvious
The antioxidation activity Acta Pharmaceutica Sinicas 2006,41 (10) of three kinds of Chinese medicines such as the shadow technology evaluation root of three-nerved spicebush:956-962.）Researchization
The antioxidation activity of compound.The compound 1H- pyrroles -2- carboxylic acids and vitamin C are configured to 2mg/ mL, take 2 μ L points
Sample carries out thin-layer chromatography in thin layer chromatography board（Solvent is chloroform：Methanol=10：1）, the lamellae spray after expansion is with 0.04%
DPPH（1,1- diphenyl -2- trinitrophenyl-hydrazines）Ethanol solution, heats 30 min at 40 DEG C, with positive control vitamin c class
Seemingly, the compound also shows white dot on blue lamellae on relevant position.The result shows that the compound pyrroles-
2- carboxylic acids have antioxidation activity（Fig. 1）, can be used for preparing anti-oxidation medicine.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, should all belong to the covering scope of the present invention.
Claims (5)
- A kind of 1. method that pyrroles's acid compound is prepared using hickory chick, it is characterised in that：Using hickory chick as raw material, through solidFermentation, the organic extraction of product, isolate and purify to obtain pyrroles's acid compound；Comprise the following steps：（1）Solid fermentation：Solid fermentation is carried out after hickory chick is activated；Then cultivated；（2）The organic extraction of product：By step（1）Obtained mycelium is cultivated to be extracted with organic solvent with culture medium；Leaching liquor is denseAfter contracting, extracted with isometric ethyl acetate and water；Ethyl acetate phase is taken, after being dehydrated, concentrating, obtains organic coarse Extract medicinal extract；（3）Isolate and purify：After organic crude extract medicinal extract is dissolved with methanol, gel filtration chromatography is carried out；Using thin-layer chromatography-biology Autography method detects the antioxidation activity of each component, after the Fraction collection with antioxidation activity, merging, then carries out anti-phase silicon Plastic column chromatography, collects the component with antioxidation activity；Normal-phase silica gel column chromatography is finally recycled, is obtained with antioxidation activity Pyrroles's acid compound；Step（1）The condition of culture is：Culture medium prescription forms：Potato 16-25wt%, glucose 1.5-2.5wt%, Peptone 0.5-1.5wt%, 1-2wt% agar, surplus is water；Wherein described hickory chick, strain are hickory chick M.co 0010（Morehellaesculenta）, July 6 in 2014 Day is preserved in Wuhan, China, and Wuhan University, China typical culture collection center, deposit number is CCTCC M 2014324；Wherein described obtained pyrroles's acid compound is that its molecular formula is C5H5NO2, structural formula is：。
- 2. the method that pyrroles's acid compound is prepared using hickory chick according to claim 1, it is characterised in that：Step（1）Its condition of the culture is nature pH, 0.1 Mpa, 100 ~ 140 DEG C of 15 ~ 40 min of sterilizing, is trained in 25-28 DEG C of constant temperature Support25-35 days.
- 3. the method according to claim 1 that pyrroles's acid compound is prepared using hickory chick, it is characterised in that：Step（2）The organic solvent is ethyl acetate, the one or more in methanol, acetone, glacial acetic acid.
- 4. the method that pyrroles's acid compound is prepared using hickory chick according to claim 1, it is characterised in that：Step（3）Its parameter of the gel filtration chromatography is：Sephadex LH-20 sephadex columns, methanol elution, flow velocity is 12 s/ Drop,Often pipe collects 5-10mL, and per test tube, sampling carries out thin-layer chromatography：Solvent is chloroform:Methanol=10:1, color developing agent is 10wt%Sulfuric acid ethanol, similar compositions merge.
- 5. the method that pyrroles's acid compound is prepared using hickory chick according to claim 1, it is characterised in that：Step（3）Its elution of the reversed-phase silica gel column chromatography is：Mixing liquid is accounted for by the use of the mixed liquor of methanol and water as eluant, eluent, methanol Long-pending5-30%, the formic acid that volume fraction is 0.1-1% is contained in mixed liquor, carries out gradient elution；The normal phase silicagel column layer AnalysisIts elution is：Eluant, eluent, volume ratio 40 are used as by the use of the mixed liquor of chloroform and methanol:1-120:1, contain body in eluant, eluent ProductFraction is the formic acid of 0.1-2 ‰.
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Non-Patent Citations (3)
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| 3种食用菌活性物质的分离纯化及结构分析;吴小君;《中国优秀硕士学位论文全文数据库农业科技辑》;20140815;第5页第1段,第6页第1段,第9页第3段,第12页第1.5节,第15页第2.4.3节,第43页第1.3节,第44-45页2.1-2.2,3.2节,第48页第3.4.3节 * |
| 应用薄层色谱-生物自显影技术评价乌药等三种中药的抗氧化性;谷丽华等;《药学学报》;20061231;第41卷(第10期);956-962 * |
| 洋来源放线菌无活性野生株的链霉素抗性抗肿瘤活性突变株新产代谢产物研究;孙玉雯等;《军事医学科学院院刊》;20100430;第34卷(第2期);摘要,第121页图1 * |
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