CN111170945B - Separation process and application of natural antioxidant amino acid derivatives in yellow mushroom - Google Patents
Separation process and application of natural antioxidant amino acid derivatives in yellow mushroom Download PDFInfo
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- C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
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Abstract
The invention relates to the technical field of separation of natural antioxidant amino acid derivatives in yellow mushrooms, in particular to a directional separation process and application of natural antioxidant amino acid derivatives in yellow mushrooms. The preparation method comprises the following steps: the method comprises four steps of extraction, enrichment of a microporous resin column, hydrophilic preparative liquid chromatography separation and hydrophilic preparative liquid chromatography purification. The invention has low cost and the product purity is more than 95 percent; the technical means adopted by the invention can be used for large-scale production: the raw material requirement is not high, the cost is low, and batch preparation is easy; the methanol is extracted by cold immersion at room temperature, and the operation is easy; the separation adopts a microporous resin column for rough separation, and the microporous resin separation material can be arranged in a medium-pressure column chromatography system and is easy to scale; the hydrophilic preparative liquid chromatography used in the separation and purification is a rapid isocratic method.
Description
Technical Field
The invention relates to the technical field of separation of natural antioxidant amino acid derivatives in yellow mushrooms, in particular to a separation process and application of natural antioxidant amino acid derivatives in yellow mushrooms.
Background
Yellow mushroom, also known as yellow green circumnuta pileus, yellow green armillaria mellea, is representative of rare wild mushroom on high mountain grasslands. The sporocarp of the product contains rich nutrients such as protein, amino acids, vitamins, minerals, volatile oil and polysaccharide, especially selenium, and is one of the best plateau treasures. Research shows that the yellow mushroom extract has better antioxidant activity. However, it is not clear what the antioxidant component is.
At present, antioxidant active components (Li Shifeng, Chenguichen and Biyurong, two wild edible fungi research on antioxidant and antitumor activities, Chinese edible fungi 2004, 24(3): 58-63) are reported to be separated from yellow mushrooms, and a multiple solvent distribution method is adopted to obtain an alcohol soluble component and a water soluble polysaccharide component with good antioxidant activity, wherein the solvent distribution frequency is 4-12 times, and the process is complicated and is not suitable for large-scale production. Chinese patents (CN 201410040146.9, CN 201410040010.8) have reported that anti-oxidation components of yellow mushroom, whose main components are nucleosides, are obtained by reverse phase solid phase extraction or preparative liquid chromatography, but no anti-oxidation active components are obtained.
At present, no literature report exists about the separation process and the application of natural antioxidant amino acid derivatives in yellow mushroom, and no systematic research is made in the existing research. Therefore, a method for separating natural antioxidant amino acid derivatives from yellow mushrooms in a simple process and large scale is needed.
Disclosure of Invention
Based on the above problems, the present invention aims to provide a process for separating natural antioxidant amino acid derivatives from yellow mushroom and the use thereof.
The separation process of natural antioxidant amino acid derivative in yellow mushroom includes the following steps:
and 4, hydrophilic preparation liquid chromatography purification: dissolving the target compound-containing component by using a methanol-water solution with the volume fraction of 0-10%, preparing a sample with the concentration of 20.0-50.0 mg/mL, filtering the sample by using a 0.45 mu m microporous filter membrane to obtain a filtrate, namely a filtrate C, purifying the filtrate C by using a hydrophilic liquid phase preparation chromatogram, detecting the purified filtrate C by using an ultraviolet detector with the detection wavelength of 254nm, collecting a main chromatographic peak fraction in the chromatographic chromatogram prepared from the filtrate C, and drying the chromatographic peak fraction under reduced pressure to obtain the antioxidant L- (+) -ergothionine with the purity of more than 95%.
Further, in the step 2, in an on-line HPLC-DPPH chromatography combined system, the first high performance liquid chromatograph adopts a zwitterionic column Click XION (250 × 4.6mm, 5 μm) hydrophilic chromatographic column, and the detection wavelength is 254 nm; and a second high performance liquid chromatograph is filled with a DPPH solution dissolved in methanol, and the detection wavelength is 517 nm.
Further, in step 1, step 3 and step 4, the drying under reduced pressure is carried out under the following conditions: the vacuum degree is 20-250 mbar, and the temperature is 40-60 ℃.
Further, in the step 2, the mobile phase A adopted by the first high performance liquid chromatograph is 0.2% formic acid-water solution, the mobile phase B is acetonitrile solution, and the flow rate of the mobile phase is 0.7-1.5 mL/min according to 0-60 min and 95-25% B; the concentration of a DPPH solution used by the second high performance liquid chromatograph is 50 mu g/mL, and the flow rate of a mobile phase is 0.4-0.5 mL/min; the length of the reaction ring is 10-18 m.
Further, in the step 3, the working parameters of the hydrophilic chromatographic column enrichment are that the length of the chromatographic column is 500mm, the diameter of the chromatographic column is 50-100 mm, and the stationary phase of the hydrophilic chromatographic column is 40-60 μm amide column XAmid, zwitterionic column Click XION or amino column NH2The mobile phase is 95-5% acetonitrile-water solution, the sample injection volume is 1-50 mL, and the flow rate is 60-300 mL/min.
Further, in the step 4, the working parameters of the hydrophilic preparative liquid chromatography are that the length of the chromatographic column is 250mm, the diameter is 20-50 mm, and the stationary phase of the hydrophilic liquid preparative column is 5 μm amide column XAmid, zwitterionic column Click XION or amino column NH2The mobile phase is acetonitrile-water solution with volume fraction of 60-90%, the sample injection volume is 2-10 mL, and the flow rate is 20-115 mL/min.
The invention also provides the application of the prepared natural antioxidant active amino acid derivative of yellow mushroom in preparing free radical scavenging medicaments or health-care foods, wherein the prepared free radical inhibitor is used as an effective component to be prepared into various medicinal preparations with any pharmaceutically acceptable carrier according to a conventional method, or is used as an effective component to be prepared into various health-care foods with any pharmaceutically acceptable carrier according to a conventional method.
Compared with the prior art, the invention has the following advantages:
(1) the invention has low cost and high product purity
The used extraction solvent, microporous resin column, reversed phase chromatographic column and hydrophilic chromatographic column can be recycled; the used chromatographic separation materials (reversed-phase preparative liquid chromatography and hydrophilic preparative liquid chromatography separation materials) can be recycled, the recycled solvent and the recycled separation materials ensure that the average cost in the separation process is low, and the two-step chromatographic separation (hydrophilic chromatographic column enrichment and hydrophilic preparative liquid chromatography purification) can ensure that the purity of the product is more than 95%.
(2) The technical means adopted by the invention can be used for large-scale production
The raw material requirement is not high, the cost is low, and the method is generally applicable to wild or market-sold yellow mushrooms and is easy for preparing materials in batches; the methanol is extracted by cold immersion at room temperature, and the operation is easy; the separation adopts a microporous resin column for rough separation, and the microporous resin separation material can be arranged in a medium-pressure column chromatography system and is easy to scale; the hydrophilic preparative liquid chromatography used in the separation and purification is a rapid isocratic method, and is very suitable for large-scale production.
Drawings
FIG. 1 is an on-line HPLC-DPPH screening chromatogram of the methanol extract of yellow mushroom of the present invention;
FIG. 2 is a diagram showing the separation of the methanol extract of yellow mushroom according to the present invention on a hydrophilic column;
FIG. 3 is a graph comparing the components of the yellow mushroom of the present invention containing the objective compound with the total sample;
FIG. 4 is a diagram of the hydrophilic preparative liquid phase separation of the target compound component of yellow mushroom according to the present invention;
FIG. 5 is a chromatogram for verifying the purity and activity of natural anti-oxidation L- (+) -ergothioneine of yellow mushroom of the present invention;
FIG. 6 is a high-resolution mass spectrum of the anti-oxidative L- (+) -ergothioneine of yellow mushroom of the present invention;
FIG. 7 shows natural anti-oxidative L- (+) -plus of yellow mushroom of the present inventionergothioneine 1H NMR nuclear magnetic map;
FIG. 8 shows the natural antioxidant L- (+) -ergothionine of yellow mushroom of the present invention 13C NMR nuclear magnetic map;
FIG. 9 is a nuclear magnetic diagram of the natural antioxidant L- (+) -ergothioneine HSQC of yellow mushroom of the present invention;
FIG. 10 is a nuclear magnetic map of the natural antioxidant L- (+) -ergothioneine HMBC of yellow mushroom of the present invention;
FIG. 11 is a planar structure diagram of the natural antioxidant L- (+) -ergothionine of yellow mushroom of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The separation process of natural antioxidant amino acid derivative in yellow mushroom includes the following steps:
And 4, hydrophilic preparation liquid chromatography purification: dissolving the target compound-containing component with 0% volume fraction methanol-water solution, preparing a sample with concentration of 50.0mg/mL, filtering with 0.45 μm microporous filter membrane to obtain a filtrate C, purifying the filtrate C by hydrophilic liquid phase preparative chromatography, detecting with an ultraviolet detector with a detection wavelength of 254nm, collecting a main chromatographic peak fraction (shown in figure 4) in a chromatogram prepared from the filtrate C, and drying the chromatographic peak fraction under reduced pressure to obtain antioxidant L- (+) -ergothionine 26.6mg with purity of more than 95% (shown in figure 5), wherein the reduced pressure drying condition is vacuum degree of 20 mbar and temperature of 40 ℃; wherein, the working parameters of the hydrophilic preparative liquid chromatography separation are that the length of a chromatographic column is 250mm, the diameter is 20 mm, the stationary phase of the hydrophilic liquid preparative column is 5 μm amide column XAmide, the mobile phase is acetonitrile-water solution with volume fraction of 90%, the sample injection volume is 5mL, and the flow rate is 20 mL/min.
The prepared natural antioxidant active amino acid derivative of yellow mushroom is applied to preparing free radical scavenging medicaments or health-care foods, wherein the prepared free radical inhibitor is used as an effective component to be prepared into various medicinal preparations with any pharmaceutically acceptable carrier according to a conventional method, or is used as an effective component to be prepared into various health-care foods with any pharmaceutically acceptable carrier according to a conventional method.
Example 2
The separation process of natural antioxidant amino acid derivative in yellow mushroom includes the following steps:
And 4, hydrophilic preparation liquid chromatography purification: dissolving the target compound-containing component by using a methanol-water solution with the volume fraction of 10%, preparing a sample with the concentration of 20.0 mg/mL, filtering the sample by using a 0.45 mu m microporous filter membrane to obtain a filtrate C, purifying the filtrate C by using a hydrophilic liquid phase preparative chromatography, detecting the purified filtrate C by using an ultraviolet detector with the detection wavelength of 254nm, collecting a main chromatographic peak fraction in a chromatogram prepared by using the filtrate C, and drying the chromatographic peak fraction under reduced pressure to obtain antioxidant L- (+) -ergothionine 132.0mg with the purity of more than 95%, wherein the reduced pressure drying condition is that the vacuum degree is 250mbar and the temperature is 60 ℃; wherein, the working parameters of the hydrophilic preparative liquid chromatography are that the length of a chromatographic column is 250mm, the diameter is 50mm, and the stationary phases of the hydrophilic preparative liquid chromatography are all 5 mu m amino column NH2The mobile phase is acetonitrile-water solution with volume fraction of 60%, the sample injection volume is 2mL, and the flow rate is 115 mL/min.
The prepared natural antioxidant active amino acid derivative of yellow mushroom is applied to preparing free radical scavenging medicaments or health-care foods, wherein the prepared free radical inhibitor is used as an effective component to be prepared into various medicinal preparations with any pharmaceutically acceptable carrier according to a conventional method, or is used as an effective component to be prepared into various health-care foods with any pharmaceutically acceptable carrier according to a conventional method.
Example 3
The separation process of natural antioxidant amino acid derivative in yellow mushroom includes the following steps:
And 4, hydrophilic preparation liquid chromatography purification: dissolving the target compound-containing component by using a methanol-water solution with the volume fraction of 5%, preparing a sample with the concentration of 40.0 mg/mL, filtering the sample by using a 0.45 mu m microporous filter membrane to obtain a filtrate C, purifying the filtrate C by using a hydrophilic liquid phase preparative chromatography, detecting the purified filtrate C by using an ultraviolet detector with the detection wavelength of 254nm, collecting a main chromatographic peak fraction in a chromatogram prepared by using the filtrate C, and drying the chromatographic peak fraction under reduced pressure to obtain antioxidant L- (+) -ergothionine 244.8mg with the purity of more than 95%, wherein the reduced pressure drying condition is that the vacuum degree is 150 mbar and the temperature is 60 ℃; the working parameters of the hydrophilic preparative liquid chromatography are that the length of a chromatographic column is 250mm, the diameter of the chromatographic column is 30 mm, the stationary phase of the hydrophilic liquid preparative column is zwitterionic column Click XION, the mobile phase is acetonitrile-water solution with volume fraction of 80%, the sample injection volume is 10mL, and the flow rate is 45 mL/min.
The prepared natural antioxidant active amino acid derivative of yellow mushroom is applied to preparing free radical scavenging medicaments or health-care foods, wherein the prepared free radical inhibitor is used as an effective component to be prepared into various medicinal preparations with any pharmaceutically acceptable carrier according to a conventional method, or is used as an effective component to be prepared into various health-care foods with any pharmaceutically acceptable carrier according to a conventional method.
Example 4
Verification of antioxidant activity of natural antioxidant L- (+) -ergothione in yellow mushroom
Adding chromatographic pure water with 2 times of the mass of the obtained natural anti-oxidation yellow mushroom into the separated natural anti-oxidation L- (+) -ergothionine for dissolving, preparing a sample with the concentration of 2 mg/mL, filtering by a 0.45-micrometer microporous filter membrane to obtain a natural anti-oxidation yellow mushroom L- (+) -ergothionine sample solution, taking 1mL of the sample, and screening the anti-oxidation activity of the yellow mushroom sample by utilizing an on-line HPLC-DPPH chromatography combined system (shown in figure 5);
in the on-line HPLC-DPPH chromatography combined system, a first high performance liquid chromatograph adopts a zwitterionic column Click XION (250 multiplied by 4.6mm, 5 mu m) hydrophilic chromatographic column, a mobile phase A adopted by the first high performance liquid chromatograph is a 0.2% formic acid-water solution, a mobile phase B is an acetonitrile solution, the flow rate of the mobile phase is 1.0 mL/min according to 0-60 min and 95-25% B, and the detection wavelength is 254 nm; a second high performance liquid chromatograph is filled with a DPPH solution dissolved in methanol, the concentration of the DPPH solution is 50 mu g/mL, and the flow rate of a mobile phase is 0.5 mL/min; the reaction ring length is 18m, the detection wavelength is 517nm, and the activity verification chromatogram is shown in figure 5.
The natural antioxidant L- (+) -ergothionine obtained from yellow mushroom has structural characteristics and structure shown in figures 6-11.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (2)
1. The separation process of the natural antioxidant amino acid derivatives in the yellow mushroom is characterized in that: the process specifically comprises the following steps:
step 1, extraction: coarsely crushing fresh yellow mushroom, and mixing the crushed materials according to a material-liquid ratio of 1 g: extracting with 5-100 mL of methanol at room temperature for 2-4 times, each time for 2-4 h, filtering, combining filtrates to obtain filtrate A, and drying the filtrate A under reduced pressure to obtain methanol extract of yellow mushroom;
step 2, enriching the microporous resin column: adding methanol with volume concentration of 70-90% and mass of 5-10 times of the methanol extract of the yellow mushroom into the methanol extract of the yellow mushroom for dissolving, preparing a sample with concentration of 50.0-100.0 mg/mL, filtering by a 0.45 mu m microporous membrane to obtain a methanol sample solution of the yellow mushroom, namely a filtrate B, taking 1mL of the filtrate B, and screening an antioxidant active ingredient in the yellow mushroom sample by using an online HPLC-DPPH (high performance liquid chromatography-DPPH (deep phase chromatography-double pH) chromatography combined system; in the on-line HPLC-DPPH chromatography combined system, a first high performance liquid chromatograph adopts a 250 x 4.6mm, 5 mu m zwitterionic column Click XION hydrophilic chromatographic column, and the detection wavelength is 254 nm; a second high performance liquid chromatograph is filled with a DPPH solution dissolved in methanol, and the detection wavelength is 517 nm; the mobile phase A adopted by the first high performance liquid chromatograph is 0.2% formic acid-water solution, the mobile phase B is acetonitrile solution, and the flow rate of the mobile phase is 0.7-1.5 mL/min according to the conditions of 0-60 min and 95-25% B; the concentration of a DPPH solution used by the second high performance liquid chromatograph is 50 mu g/mL, and the flow rate of a mobile phase is 0.4-0.5 mL/min; the length of the reaction ring is 10-18 m;
step 3, hydrophilic preparative liquid chromatography separation: separating the filtrate B with hydrophilic chromatographic column, detecting with ultraviolet detector with detection wavelength of 254nm, collecting main chromatographic peak fraction in the preparative chromatogram, and drying under reduced pressure to obtain the final product containing target compoundA component of (a); the working parameters of the enrichment of the hydrophilic chromatographic column are that the length of the chromatographic column is 500mm, the diameter of the chromatographic column is 50-100 mm, and the stationary phase of the hydrophilic chromatographic column is amide column XAmide of 40-60 mu m, zwitterionic column ClickXION or amino column NH2The mobile phase is 95-5% acetonitrile-water solution, the sample injection volume is 1-50 mL, and the flow rate is 60-300 mL/min;
and 4, hydrophilic preparation liquid chromatography purification: dissolving the target compound-containing component by using a methanol-water solution with the volume fraction of 0-10%, preparing a sample with the concentration of 20.0-50.0 mg/mL, filtering the sample by using a 0.45 mu m microporous filter membrane to obtain a filtrate, namely a filtrate C, purifying the filtrate C by using a hydrophilic liquid phase preparation chromatogram, detecting the purified filtrate C by using an ultraviolet detector with the detection wavelength of 254nm, collecting a main chromatographic peak fraction in a chromatogram prepared by using the filtrate C, and drying the chromatographic peak fraction under reduced pressure to obtain the antioxidant L- (+) -ergothionine with the purity of more than 95%; the working parameters of the hydrophilic preparative liquid chromatography separation are that the length of a chromatographic column is 250mm, the diameter of the chromatographic column is 20-50 mm, and the stationary phase of the hydrophilic preparative liquid chromatography column is 5 mu m amide column XAmide, zwitterionic column Click XION or amino column NH2The mobile phase is acetonitrile-water solution with volume fraction of 60-90%, the sample injection volume is 2-10 mL, and the flow rate is 20-115 mL/min.
2. The process of claim 1 for the isolation of natural antioxidant amino acid derivatives from yellow mushrooms, wherein: in the step 1, the step 3 and the step 4, the conditions of reduced pressure drying are as follows: the vacuum degree is 20-250 mbar, and the temperature is 40-60 ℃.
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