CN104450818A - Method for preparing pyrrole acid compound by using morehella esculenta and application of pyrrole acid compound - Google Patents
Method for preparing pyrrole acid compound by using morehella esculenta and application of pyrrole acid compound Download PDFInfo
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- CN104450818A CN104450818A CN201410679109.2A CN201410679109A CN104450818A CN 104450818 A CN104450818 A CN 104450818A CN 201410679109 A CN201410679109 A CN 201410679109A CN 104450818 A CN104450818 A CN 104450818A
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Abstract
The invention belongs to the field of separation and extraction of natural medicine, and particularly relates to a method for preparing a pyrrole acid compound by using morehella esculenta and an application of the pyrrole acid compound. The specific preparation method is as follows: with morehella esculenta as a raw material, the pyrrole acid compound is obtained by solid fermentation, organic product extraction, separation and purification. The used morehella esculenta raw material is M.co0010 (Morehella esculenta), which has been preserved in the China Center for Type Culture Collection, Chinese Wuhan University on July 6, 2014, and the preservation number is CCTCC NO: M2014324. The pyrrole acid compound is obtained according to the preparation method, and experiments prove that the pyrrole acid compound has antioxidant activity and can be used for preparing antioxidant drugs. The pyrrole acid compound prepared in the invention has better antioxidant activity and can be prepared by fermenting a strain (i) Morehellaesculenta (/i), the raw material resources of a culture medium are wide, the preparation method is simple, and industrialized production is easy to achieve.
Description
Technical field
The invention belongs to natural drug separation and Extraction field, be specifically related to a kind of utilize morel to prepare minaline compound method and application.
Background technology
Natural Antioxidants has scavenging free radicals, and then has effect that is anti-ageing and the various disease of prevention.Edible medicinal macro fungi is not only rich in the nutritive substances such as amino acid, VITAMIN, polysaccharide, protein, and its secondary metabolite structure type is various, these secondary metabolisms have anti-oxidant, antibacterium, the synthesis of promotion nerve growth factor, enzyme inhibitors, antimycotic, the multiple biological activity such as cell toxicant, receptor antagonist.Anti-oxidant function is one of critical function of edible medicinal fungus, and separation and Extraction Natural Antioxidants from edible medicinal fungus, contributes to the exploitation of edible medicinal fungus resource, has enriched natural pharmaceutical resources, promotes human health development and disease prevention.Based on above background, carry out research of the present invention.
Summary of the invention
The object of the invention is to contain abundant Natural Antioxidants in edible mushrooms, but development and utilization level is low; There is provided a kind of utilize morel to prepare minaline compound method and application.Minaline compound prepared by the present invention has good anti-oxidant activity, and culture medium raw material wide material sources, preparation method is simple, easily realizes suitability for industrialized production.
For achieving the above object, the present invention adopts following technical scheme:
A kind of morel, described bacterial classification is morel M.co 0010(Morehella esculenta), be preserved in Wuhan, China Wuhan University China typical culture collection center on July 6th, 2014, deposit number is CCTCC M 2014324.
Utilizing morel to prepare a method for minaline compound, take morel as raw material, obtains minaline compound through solid fermentation, the organic lixiviate of product, separation and purification.
The described morel that utilizes prepares the method for minaline compound, comprises the following steps:
(1) solid fermentation: carry out solid fermentation after being activated by morel; Then cultivate;
(2) the organic lixiviate of product: step (1) is cultivated the mycelium obtained and the lixiviate of substratum organic solvent; After vat liquor is concentrated, extract by isopyknic ethyl acetate and water; Get ethyl acetate phase, after dehydration, concentrating, obtain organic coarse extract medicinal extract;
(3) separation and purification: after organic coarse extract medicinal extract dissolve with methanol, carry out gel filtration chromatography; Employing thin-layer chromatography-bioautography (reference: Gu Lihua etc. the anti-oxidant activity of three kinds of Chinese medicines such as the application TLC-bioautography technological assessment root of three-nerved spicebush. Acta Pharmaceutica Sinica. 2006,41 (10): 956-962) anti-oxidant activity of each component is detected, by have anti-oxidant activity Fraction collection, merge after, carry out reversed-phase silica gel column chromatography again, collect the component with anti-oxidant activity; Finally recycle purification on normal-phase silica gel column chromatography, obtain the minaline compound with anti-oxidant activity.
The culture medium prescription of its condition solid fermentation of cultivation described in step (1) consists of: potato 16-25wt%, glucose 1.5-2.5wt%, peptone 0.5-1.5wt%, 1-2wt% agar, and surplus is water; Nature pH, 0.1 Mpa, 100 ~ 140 DEG C of sterilizing 15 ~ 40 min, in 25-28 DEG C of constant temperature culture 25-35 days.
Organic solvent described in step (2) is one or more in ethyl acetate, methyl alcohol, acetone, glacial acetic acid.
Its parameter of gel filtration chromatography described in step (3) is: Sephadex LH-20 sephadex column, methanol-eluted fractions, flow velocity is that 12 s/ drip, often 5-10mL collected by pipe, thin-layer chromatography is carried out in every test tube sampling: developping agent is chloroform: methyl alcohol=10:1, developer is the sulfuric acid ethanol of 10wt%, and similar compositions merges.
Its wash-out of reversed-phase silica gel column chromatography described in step (3) is: use the mixed solution of methyl alcohol and water as eluent, methyl alcohol accounts for the 5-30% of mixeding liquid volume, and containing volume fraction in mixed solution is the formic acid of 0.1-1%, carries out gradient elution; Described its wash-out of purification on normal-phase silica gel column chromatography is: use the mixed solution of chloroform and methyl alcohol as eluent, volume ratio is 40:1-120:1, is the formic acid of 0.1-2 ‰ in eluent containing volume fraction.
The minaline compound that method as above obtains, its molecular formula is C
5h
5nO
2, structural formula is:
.
The application of described minaline compound, is specially: for the preparation of anti-oxidation medicine.
Beneficial effect of the present invention is:
Minaline compound prepared by the present invention has good anti-oxidant activity, can utilize bacterial strain
morehella esculentafermentation obtains, culture medium raw material wide material sources, and preparation method is simple, easily realizes suitability for industrialized production.
Accompanying drawing explanation
The thin-layer chromatogram of Fig. 1 compound pyrroles-2-carboxylic acid; 1 represents contrast vitamins C, and 2 represent compound pyrroles-2-carboxylic acid.
Embodiment
The present invention's the following example further illustrates the present invention, but protection scope of the present invention is not limited to the following example.
Minaline compound provided by the invention preparation method, the steps include:
1) solid fermentation and the organic lixiviate of product: by morel (
morehella esculenta) slant strains first activates on culture plate, culture temperature 26 DEG C, incubation time 10 days, then carry out large batch of solid fermentation, employing culture medium prescription is: containing potato 200 g in every premium on currency, glucose 20 g, peptone 10 g, agar 10 g, pH nature, 0.1 Mpa, 100 ~ 140 DEG C of sterilizing 15 ~ 40 min, are placed in 25-28 DEG C of constant temperature culture.Ferment after 25-35 days, by centrifuging by mycelium and separation of fermentative broth.Mycelium and substratum ethyl acetate, methyl alcohol, acetone and other organic solvent lixiviate after cultivating.Extract with ethyl acetate and water equal-volume after vat liquor is concentrated, concentrated after ethyl acetate phase dehydration, obtain organic coarse extract medicinal extract.
2) separating-purifying: by 1) described in organic coarse extract medicinal extract dissolve with methanol, Sephadex LH-20 dextrane gel column chromatography, methanol-eluted fractions, flow velocity is about 12 s/ and drips, and often 5-10 mL collected by pipe.Thin-layer chromatography-bioautography is adopted to detect the anti-oxidant activity of often pipe, merge the activated component of tool, with dissolve with methanol, carry out reversed-phase silica gel column chromatography, with containing volume fraction be the mixed solution of the methyl alcohol of 0.1-1% formic acid and water as eluent, methyl alcohol accounts for the 5-30% of mixeding liquid volume, carries out gradient elution, be in charge of collection, merge the component of anti-oxidant activity; Finally utilize purification on normal-phase silica gel chromatography, with containing volume fraction be the mixed solution of the chloroform of 0.1-2 ‰ formic acid and methyl alcohol as eluent, volume ratio is 40:1-120:1, obtains the minaline pure compounds with anti-oxidant activity.
X-ray single crystal diffraction and nuclear magnetic resonance spectroscopy are carried out to sterling, and carries out anti-oxidant activity test.
According to X-ray single crystal diffraction and nuclear magnetic resonance data, Structural Identification is carried out to compound, can the structure of deterministic compound.
Compound of the present invention is minaline compound pyrroles-2-carboxylic acid, and molecular formula is C
5h
5nO
2, structural formula is as follows:
。
According to the Antioxidative Activity Determination of compound pyrroles-2-carboxylic acid, find that pyrroles-2-carboxylic acid has stronger anti-oxidant activity (embodiment 3).
Embodiment 1
With potato glucose solid medium (containing potato 200 g, glucose 20 g, peptone 10 g in every premium on currency, agar 10 g, pH nature, 0.1 MPa, 121 DEG C of sterilizing 30 min), by the bacterial strain after activation
morehella esculentasolid culture 9.6 L, cultivates 30 d in 26 DEG C of shaking tables.Mycelium and substratum ethyl acetate after cultivating: methyl alcohol: glacial acetic acid volume ratio is the organic solvent lixiviate of 80:15:5.Extract with ethyl acetate and water equal-volume after vat liquor is concentrated, concentrated after ethyl acetate phase dehydration, obtain methanol soluble crude extract (1.54 g, brown medicinal extract).
By 1.54 g methanol crude extract in previous step, fully dissolve by proper amount of methanol, carry out Sephadex LH-20 dextrane gel column chromatography, methanol-eluted fractions, flow velocity is about 12 s/ and drips, often 5mL collected by pipe, thin-layer chromatography is carried out in every test tube sampling, and (developping agent is chloroform: methyl alcohol=10:1, developer: 10% sulfuric acid ethanol) analyze, similar compositions merges, and obtains 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.Adopt thin-layer chromatography-bioautography to detect the anti-oxidant activity of every component, obtain the component F (240.4 mg) with anti-oxidant activity.Reverse phase silica gel post (30 g) chromatography, obtain F1.1(16.7 mg with 400 ml 30% methanol-water wash-outs) and F1.2(11.7 mg are carried out to component F).Component F1.1 recycles Sephadex LH-20 sephadex column layer, and with the acetone wash-out containing 1 ‰ formic acid, flow velocity is about 12 s/ and drips, and often 2.5 mL collected by pipe, obtains F1.1.1(8 mg).Component F1.1.1 finally utilizes purification on normal-phase silica gel chromatography 80 ml chloroform-methanol (80:1, containing 1 ‰ formic acid in chloroform) wash-out to obtain the described pure compounds (1.5 mg) with anti-oxidant activity.
The structure of compound determines the method that can adopt X-ray single crystal diffraction.According to the X-ray single crystal diffraction data of described compound, the structure of described compound can be determined.
The X-ray single crystal diffraction data of described compound: monocline day crystallographic system, crystallographic dimension: 0.7 × 0.7 × 0.4 mm, spacer is C 2/c, unit cell parameters:
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
-30m
3,
z=4,
dc=1.507 g cm
-3,
μ(Mo
kα)=0.119 ㎜
-1, solved by direct method, use method of least squares refine, the structure of described compound is determined.
Embodiment 2
Similar to Example 1, its difference be Identification of chemical structure adopt NMR (Nuclear Magnetic Resonance) spectrum (
1h-NMR,
13c-NMR, HSQC, HMBC,
1h,
1h-COSY) analytic method, can the structure of deterministic compound according to NMR data.
Described compound
1h NMR [500 MHz, (CD
3)
2cO]: δ 6.85 (m, 1H, H-3), δ 6.21 (m, 1H, H-4), δ 7.05 (m, 1H, H-5).Described compound
13c NMR [500 MHz, (CD
3)
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 compound
1h-NMR and
13the chemical displacement value of C-NMR shows in compound containing mono-substituted pyrrole structure unit.According to another
13c-NMR chemical displacement value δ 162.0 s infers that it is the carboxyl being connected in pyrrole ring No. 2 positions.
Embodiment 3
Adopt thin-layer chromatography-bioautography (reference: Gu Lihua, Deng. the anti-oxidant activity of three kinds of Chinese medicines such as the application TLC-bioautography technological assessment root of three-nerved spicebush. Acta Pharmaceutica Sinica. 2006,41 (10): 956-962.) study the anti-oxidant activity of compound.Described compound 1H-pyrroles-2-carboxylic acid and vitamins C are mixed with 2mg/ mL, get 2 μ L point samples in thin layer chromatography board, carry out thin-layer chromatography (developping agent is chloroform: methyl alcohol=10:1), thin layer plate spray after expansion is with 0.04% DPPH(1,1-phenylbenzene-2-trinitrophenyl-hydrazine) ethanolic soln, at 40 DEG C, heat 30 min, with positive control vitamin c class seemingly, described compound also aobvious white dot on corresponding position on blue thin layer plate.Result shows that described compound pyrroles-2-carboxylic acid has anti-oxidant activity (Fig. 1), may be used for preparing anti-oxidation medicine.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (9)
1. utilize morel to prepare a method for minaline compound, it is characterized in that: take morel as raw material, obtain minaline compound through solid fermentation, the organic lixiviate of product, separation and purification.
2. the method utilizing morel to prepare minaline compound according to claim 1, is characterized in that: comprise the following steps:
(1) solid fermentation: carry out solid fermentation after being activated by morel; Then cultivate;
(2) the organic lixiviate of product: step (1) is cultivated the mycelium obtained and the lixiviate of substratum organic solvent; After vat liquor is concentrated, extract by isopyknic ethyl acetate and water; Get ethyl acetate phase, after dehydration, concentrating, obtain organic coarse extract medicinal extract;
(3) separation and purification: after organic coarse extract medicinal extract dissolve with methanol, carry out gel filtration chromatography; Adopt thin-layer chromatography-bioautography to detect the anti-oxidant activity of each component, by have anti-oxidant activity Fraction collection, merge after, then carry out reversed-phase silica gel column chromatography, collect the component with anti-oxidant activity; Finally recycle purification on normal-phase silica gel column chromatography, obtain the minaline compound with anti-oxidant activity.
3. the method utilizing morel to prepare minaline compound according to claim 2, it is characterized in that: the culture condition described in step (1) is: culture medium prescription consists of: potato 16-25wt%, glucose 1.5-2.5wt%, peptone 0.5-1.5wt%, 1-2wt% agar, surplus is water.
4. the method utilizing morel to prepare minaline compound according to claim 2, is characterized in that: its condition of cultivation described in step (1) is nature pH, and 0.1 Mpa, 100 ~ 140 DEG C of sterilizing 15 ~ 40 min, in 25-28 DEG C of constant temperature culture 25-35 days.
5. the method utilizing morel to prepare minaline compound according to claim 2, is characterized in that: the organic solvent described in step (2) is one or more in ethyl acetate, methyl alcohol, acetone, glacial acetic acid.
6. the method utilizing morel to prepare minaline compound according to claim 2, it is characterized in that: its parameter of gel filtration chromatography described in step (3) is: Sephadex LH-20 sephadex column, methanol-eluted fractions, flow velocity is that 12 s/ drip, often 5-10mL collected by pipe, thin-layer chromatography is carried out in every test tube sampling: developping agent is chloroform: methyl alcohol=10:1, and developer is the sulfuric acid ethanol of 10wt%, and similar compositions merges.
7. the method utilizing morel to prepare minaline compound according to claim 2, it is characterized in that: its wash-out of reversed-phase silica gel column chromatography described in step (3) is: use the mixed solution of methyl alcohol and water as eluent, methyl alcohol accounts for the 5-30% of mixeding liquid volume, containing volume fraction in mixed solution is the formic acid of 0.1-1%, carries out gradient elution; Described its wash-out of purification on normal-phase silica gel column chromatography is: use the mixed solution of chloroform and methyl alcohol as eluent, volume ratio is 40:1-120:1, is the formic acid of 0.1-2 ‰ in eluent containing volume fraction.
8. the minaline compound that obtains of method as claimed in claim 1 or 2, is characterized in that: described its molecular formula of minaline compound is C
5h
5nO
2, structural formula is:
.
9. an application for minaline compound as claimed in claim 8, is characterized in that: for the preparation of anti-oxidation medicine.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115154490A (en) * | 2022-07-22 | 2022-10-11 | 四川省食用菌研究所 | Antibacterial active site of morchella sporocarp and preparation method thereof |
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| 吴小君: "3种食用菌活性物质的分离纯化及结构分析", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
| 孙玉雯等: "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", 《军事医学科学院院刊》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115154490A (en) * | 2022-07-22 | 2022-10-11 | 四川省食用菌研究所 | Antibacterial active site of morchella sporocarp and preparation method thereof |
| CN115154490B (en) * | 2022-07-22 | 2023-11-21 | 四川省食用菌研究所 | Morchella fruiting body antibacterial active site and its preparation method |
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Effective date of registration: 20210611 Address after: 362417 No.17, Tianshou Fudi Road, kangsui village, Taozhou Township, Anxi County, Quanzhou City, Fujian Province Patentee after: ANXI TAOYUAN ORGANIC TEA FARM Co.,Ltd. Address before: 350117 Qishan campus of Fujian Normal University, Fuzhou University Town, Fujian Province Patentee before: Fujian Normal University |