CN104450818B - A kind of method and its application that pyrroles's acid compound is prepared using hickory chick - Google Patents

Abstract

The invention belongs to the field of separation and extraction of natural medicines, and in particular relates to a method for preparing pyrrole acid compounds by using morels and an application thereof. The specific preparation method is as follows: taking hickory chick as a raw material, obtaining the pyrrole acid compound through solid fermentation, organic extraction of the product, separation and purification. The raw material of morel used is morehella esculenta M.co 0010 (Morehella esculenta), which has been preserved in the Chinese Type Culture Collection Center of Wuhan University, Wuhan, China on July 6, 2014, with the preservation number CCTCC M 2014324. According to the preparation method, a pyrrole acid compound is obtained, and experiments prove that the pyrrole acid compound has antioxidant activity and can be used to prepare antioxidant drugs. The pyrrole acid compound prepared by the invention has good antioxidant activity, can be prepared by fermentation of the strain Morehella esculenta , has wide sources of culture medium raw materials, simple preparation method, and is easy to realize industrialized production.

Description

A kind of method utilizing morel to prepare pyrrole acid compound and its application

technical field

The invention belongs to the field of separation and extraction of natural medicines, and in particular relates to a method for preparing pyrrole acid compounds by using morels and an application thereof.

Background technique

Natural antioxidant substances can scavenge free radicals, and then have the effect of anti-aging and preventing various diseases. Edible and medicinal macrofungi are not only rich in nutrients such as amino acids, vitamins, polysaccharides, and proteins, but also have various types of secondary metabolites. Fungal, cytotoxic, receptor antagonist and other biological activities. Antioxidant function is one of the important functions of edible and medicinal fungi. The isolation and extraction of natural antioxidant substances from edible and medicinal fungi will help the development and utilization of edible and medicinal fungus resources, enrich natural drug resources, and promote human health and development. prevent disease. Based on the above background, carry out research of the present invention.

Contents of the invention

The purpose of the present invention is to provide a method for preparing pyrrole acid compounds by using Morchella fungus and its application against the fact that edible fungi are rich in natural antioxidant substances, but the degree of development and utilization is low. The pyrrole acid compound prepared by the invention has better antioxidant activity, and the source of culture medium raw materials is wide, the preparation method is simple, and industrial production can be easily realized.

To achieve the above object, the present invention adopts the following technical solutions:

Morehella esculenta, the strain of which is M.co 0010 (Morehella esculenta), has been preserved in the Chinese Type Culture Collection Center of Wuhan University, Wuhan, China on July 6, 2014, and the preservation number is CCTCC M2014324.

The invention discloses a method for preparing pyrrolic acid compounds by using morels. The morels are used as raw materials to obtain the pyrrolic acid compounds through solid fermentation, organic extraction of products, and separation and purification.

The method for preparing the pyrrole acid compound by using Morchella, comprises the following steps:

(1) Solid fermentation: activate morels and carry out solid fermentation; then culture;

(2) Organic extraction of the product: extract the mycelia and culture medium obtained in step (1) with an organic solvent; after the extraction solution is concentrated, extract it with an equal volume of ethyl acetate and water; take ethyl acetate phase, after dehydration and concentration, the organic crude extract extract is obtained;

(3) Separation and purification: After dissolving the crude organic extract extract in methanol, perform gel column chromatography; use thin-layer chromatography-bioautographic method (reference: Gu Lihua et al. The development technique was used to evaluate the antioxidant activity of three traditional Chinese medicines such as Wuyao. Acta Pharmaceutica Sinica. 2006, 41(10):956-962) to detect the antioxidant activity of each component, and after the components with antioxidant activity were collected and combined, Reverse-phase silica gel column chromatography is then performed to collect components with antioxidant activity; finally, normal-phase silica gel column chromatography is used to obtain the pyrrolic acid compound with antioxidant activity.

The culture medium formula of the conditional solid fermentation described in step (1) consists of: potato 16-25wt%, glucose 1.5-2.5wt%, peptone 0.5-1.5wt%, 1-2wt% agar, and the balance is water; Natural pH, 0.1 Mpa, sterilized at 100-140°C for 15-40 minutes, and cultured at 25-28°C for 25-35 days.

The organic solvent described in step (2) is one or more of ethyl acetate, methanol, acetone, and glacial acetic acid.

The parameters of the gel column chromatography described in step (3) are: Sephadex LH-20 dextran gel column, eluted with methanol, the flow rate is 12 s/drop, 5-10mL is collected in each tube, and each tube is sampled for thin Layer chromatography: The developing agent is chloroform:methanol=10:1, the color developing agent is 10wt% ethanol sulfate, and similar components are combined.

The elution of the reversed-phase silica gel column chromatography described in step (3) is: use a mixture of methanol and water as the eluent, methanol accounts for 5-30% of the volume of the mixture, and the mixture contains a volume fraction of 0.1- 1% formic acid for gradient elution; the elution of the normal phase silica gel column chromatography is as follows: use a mixture of chloroform and methanol as the eluent, the volume ratio is 40:1-120:1, the eluent Contains formic acid with a volume fraction of 0.1-2‰.

A kind of pyrrole acid compound obtained by the above method, its molecular formula is C ₅ H ₅ NO ₂ , and its structural formula is:

.

The application of the pyrrole acid compound is specifically: for the preparation of antioxidant drugs.

The beneficial effects of the present invention are:

The pyrrole acid compound prepared by the invention has good antioxidant activity, can be prepared by fermentation of the strain Morehella esculenta , has wide sources of culture medium raw materials, simple preparation method, and is easy to realize industrialized production.

Description of drawings

Fig. 1 is the thin-layer chromatogram of compound pyrrole-2-carboxylic acid; 1 represents the control vitamin C, and 2 represents compound pyrrole-2-carboxylic acid.

Detailed ways

The present invention further illustrates the present invention with following examples, but protection scope of the present invention is not limited to following examples.

The preparation method of pyrrole acid compound provided by the invention, its steps are:

1) Solid fermentation and organic extraction of products: first activate Morehella esculenta slant strains on a culture plate, culture temperature 26 ℃, culture time 10 days, and then carry out large-scale solid fermentation, using the culture medium formula Contains 200 g of potatoes, 20 g of glucose, 10 g of peptone, 10 g of agar per liter of water, natural pH, 0.1 Mpa, sterilized at 100-140°C for 15-40 min, and cultured at a constant temperature of 25-28°C. After 25-35 days of fermentation, the mycelium is separated from the fermentation broth by centrifugation. After cultivation, the mycelia and medium are extracted with organic solvents such as ethyl acetate, methanol, and acetone. The extract is concentrated and extracted with equal volumes of ethyl acetate and water, and the ethyl acetate phase is dehydrated and then concentrated to obtain the crude organic extract.

2) Separation and purification: dissolve the crude organic extract described in 1) with methanol, perform Sephadex LH-20 dextran gel column chromatography, elute with methanol, the flow rate is about 12 s/drop, and collect 5 -10 mL. The antioxidant activity of each tube was detected by thin-layer chromatography-bioautographic method, and the active components were combined, dissolved in methanol, and subjected to reverse-phase silica gel column chromatography, with methanol containing 0.1-1% formic acid and The mixed solution of water is used as the eluent, methanol accounts for 5-30% of the volume of the mixed solution, gradient elution is performed, collected in separate tubes, and the components with antioxidant activity are combined; finally, normal phase silica gel chromatography is used, with a volume fraction of 0.1 -2‰ formic acid mixed solution of chloroform and methanol is used as eluent, the volume ratio is 40:1-120:1, and the pure product of pyrrole acid compound with antioxidant activity is obtained.

The pure product was analyzed by X-ray single crystal diffraction and nuclear magnetic resonance, and the antioxidant activity was tested.

According to X-ray single crystal diffraction and nuclear magnetic resonance data, the structure of the compound is identified, and the structure of the compound can be determined.

The compound described in the present invention is pyrrole acid compound pyrrole-2-carboxylic acid, the molecular formula is C ₅ H ₅ NO ₂ , and the structural formula is as follows:

.

According to the determination of the antioxidant activity of the compound pyrrole-2-carboxylic acid, it was found that pyrrole-2-carboxylic acid has strong antioxidant activity (Example 3).

Example 1

The activated strain Morehella esculenta was cultured on solid medium with potato dextrose (200 g potato, 20 g glucose, 10 g peptone, 10 g agar per liter of water, natural pH, 0.1 MPa, sterilized at 121 °C for 30 min). 9.6 L, cultured on a shaker at 26 °C for 30 days. After cultivation, the mycelia and the culture medium are extracted with an organic solvent with a volume ratio of ethyl acetate:methanol:glacial acetic acid of 80:15:5. The extract was concentrated and extracted with equal volumes of ethyl acetate and water, and the ethyl acetate phase was dehydrated and concentrated to obtain methanol-soluble crude extract (1.54 g, brown extract).

Dissolve 1.54 g of the crude methanol extract in the previous step with an appropriate amount of methanol, perform Sephadex LH-20 Sephadex column chromatography, and elute with methanol at a flow rate of about 12 s/drop. Test tube samples were taken for thin layer chromatography (developing solvent: chloroform:methanol=10:1, chromogenic agent: 10% sulfuric acid ethanol) analysis, similar components were combined to 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) components. The antioxidant activity of each component was detected by TLC-bioautographic method, and the component F (240.4 mg) with antioxidant activity was obtained. Fraction F was subjected to reverse-phase silica gel column (30 g) chromatography and eluted with 400 ml of 30% methanol water to obtain F1.1 (16.7 mg) and F1.2 (11.7 mg). Component F1.1 was then eluted with Sephadex LH-20 sephadex column layer with acetone containing 1‰ formic acid, the flow rate was about 12 s/drop, and 2.5 mL was collected in each tube to obtain F1.1.1 (8 mg ). Component F1.1.1 was finally eluted by normal phase silica gel chromatography with 80 ml of chloroform-methanol (80:1, containing 1‰ formic acid in chloroform) to obtain the pure compound (1.5 mg) with antioxidant activity.

The structure of the compound can be determined by X-ray single crystal diffraction method. According to the X-ray single crystal diffraction data of the compound, the structure of the compound can be determined.

X-ray single crystal diffraction data of the compound: monoclinic heliotropic system, crystal size: 0.7×0.7×0.4 mm, space group C 2/c, unit cell parameter: a =13.2599 (14)×10 ^-10 m , b = 5.0253(5) ×10 ^-10 m, c =14.8921(16) ×10 ^-10 m, α=90°, β=99.199(11) °, γ=90°, V =979.57(18) × 10 ^-30 m ³ , Z =4, Dc = 1.507 g∙cm ^-3 , μ (Mo K α) = 0.119㎜ ^-1 , solved by direct method and refined by least square method, the structure of the compound is obtained Sure.

Example 2

Similar to Example 1, the difference is that the chemical structure identification adopts the analysis method of nuclear magnetic resonance ( ¹ H-NMR, ¹³ C-NMR, HSQC, HMBC, ¹ H, ¹ H-COSY), and the identity of the compound can be determined according to the NMR data. structure.

¹ H NMR [500 MHz, (CD ₃ ) ₂ CO] of the compound: δ 6.85 (m, 1H, H-3), δ 6.21 (m, 1H, H-4), δ 7.05 (m, 1H, H-5). ¹³ C NMR [500 MHz, (CD ₃ ) ₂ OD] of the compound: 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). The chemical shift values of ¹ H-NMR and ¹³ C-NMR of the compound indicate that the compound contains a monosubstituted pyrrole structural unit. According to ^{the 13} C-NMR chemical shift value δ162.0 s, it is presumed that it is the carboxyl group attached to the 2-position of the pyrrole ring.

Example 3

Using thin-layer chromatography-bioautoradiography (reference: Gu Lihua, et al. Application of thin-layer chromatography-bioautoradiography to evaluate the antioxidant activity of three traditional Chinese medicines such as black medicine. Acta Pharmaceutica Sinica. 2006, 41(10): 956-962.) to study the antioxidant activity of compounds. The compound 1H-pyrrole-2-carboxylic acid and vitamin C were formulated to 2 mg/mL, and 2 μL was spotted on a thin-layer chromatography plate for thin-layer chromatography (developing agent: chloroform:methanol=10:1 ), the developed TLC plate was sprayed with 0.04% DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) ethanol solution, heated at 40 °C for 30 min, similar to the positive control vitamin C, the The compound also showed white spots on the corresponding position on the blue TLC plate. The results show that the compound pyrrole-2-carboxylic acid has antioxidant activity (Figure 1), and can be used to prepare antioxidant drugs.

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims (5)

1. a method utilizing hickory chick to prepare pyrrole acid compound is characterized in that: taking hickory chick as raw material, through solid Fermentation, product organic extraction, separation and purification to obtain the pyrrole acid compound; including the following steps: (1) Solid fermentation: activate morels and carry out solid fermentation; then culture; (2) Organic extraction of the product: Extract the mycelium and culture medium obtained in step (1) with an organic solvent; the extraction solution is concentrated After shrinking, extract with equal volumes of ethyl acetate and water; take the ethyl acetate phase, dehydrate and concentrate to obtain the organic crude extract; (3) Separation and purification: After dissolving the crude organic extract extract in methanol, gel column chromatography was performed; the antioxidant activity of each component was detected by thin-layer chromatography-bioautographic method, and the antioxidant activity After the components are collected and combined, reverse-phase silica gel column chromatography is performed to collect components with antioxidant activity; finally, normal-phase silica gel column chromatography is used to obtain the pyrrole acid compound with antioxidant activity; The culture conditions described in step (1) are: the composition of the culture medium is: 16-25wt% potato, 1.5-2.5wt% glucose, 0.5-1.5wt% peptone, 1-2wt% agar, and the balance is water; Among them, the morel, the strain of which is Morchella M.co 0010 (Morehella esculenta), has been preserved in Wuhan, China, Wuhan University, China Type Culture Collection Center on July 6, 2014, and the preservation number is CCTCC M 2014324 ; Wherein the obtained pyrrole acid compound has a molecular formula of C ₅ H ₅ NO ₂ and a structural formula of: . 2. The method for preparing pyrrolic acid compounds from Morchella according to claim 1, characterized in that: step (1) The conditions of the culture are natural pH, 0.1 Mpa, 100-140°C sterilization for 15-40 min, and constant temperature cultivation at 25-28°C 25-35 days. 3. The method for preparing pyrrolic acid compounds by using Morchella according to claim 1, characterized in that: step (2) The organic solvent is one or more of ethyl acetate, methanol, acetone, and glacial acetic acid. 4. The method for preparing pyrrolic acid compounds from Morchella according to claim 1, characterized in that: step (3) Its parameter of described gel column chromatography is: Sephadex LH-20 dextran gel column, methanol elution, flow velocity is 12 s/ drop, Collect 5-10mL in each tube, take samples from each test tube for thin-layer chromatography: the developing solvent is chloroform:methanol=10:1, and the chromogenic reagent is 10wt% The sulfuric acid ethanol, similar components are combined. 5. The method for preparing pyrrolic acid compounds from Morchella according to claim 1, characterized in that: step (3) Its elution of described reverse phase silica gel column chromatography is: use the mixed solution of methanol and water as eluent, methanol accounts for the volume of mixed solution 5-30%, the mixed solution contains formic acid with a volume fraction of 0.1-1%, and gradient elution is carried out; the normal phase silica gel column chromatography Its elution is: use a mixture of chloroform and methanol as the eluent, the volume ratio is 40:1-120:1, and the eluent contains volume Formic acid with a fraction of 0.1-2‰.