CN114276396A - N having pancreatic lipase inhibitory activity6-ethyl acetate-3' -acetyl-beta-ribose adenosine and preparation method thereof - Google Patents
N having pancreatic lipase inhibitory activity6-ethyl acetate-3' -acetyl-beta-ribose adenosine and preparation method thereof Download PDFInfo
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Abstract
The invention relates to N with the function of inhibiting pancreatic lipase activity6An ethyl acetate-3' -ethyl acetate-beta-ribose adenosine and a preparation method thereof, belonging to the technical field of extraction and preparation of biological products. N is a radical of6The-ethyl acetate-3' -ethyl ester-beta-ribose adenosine is a yellow oily substance, and N is respectively obtained by one-dimensional and two-dimensional nuclear magnetic resonance and accurate mass spectrum identification6-ethyl acetate-3' -ethyl ester-beta-ribose adenosine structural formula. N of the invention6The-ethyl acetate-3' -ethyl ester-beta-ribose adenosine is obtained by culturing cordyceps sobolifera fungus and then extracting and separating. N of the invention6-BThe acid carbethoxy-3' -carbethoxy-beta-riboadenosine can be used for preparing pancreatic lipase inhibitors, has the potential of treating obesity, and also has potential effect on diabetes. The preparation method of the invention adopts microbial fermentation production, is environment-friendly, is not influenced by natural environment and resources, and is easy to realize industrialized, automatic and continuous production.
Description
Technical Field
The invention belongs to the technical field of extraction and preparation of biological products, and particularly relates to N with pancreatic lipase activity inhibition function6Extraction, purification, structural identification and activity determination of-ethyl acetate-3' -acetyl-beta-ribose adenosine.
Background
N6The-ethyl acetate-5' -acetyl-beta-ribose adenosine is a novel bioactive substance which is separated and prepared from a culture of cicada fungus (Cordyceps chanhua) and has the function of inhibiting the activity of pancreatic lipase enzyme.
Cordyceps sobolifera (Cordyceps chanhua) is a fungus of Clavicipitaceae (Clavicipitaceae) and Cordyceps (Cordyceps) and has multiple synonyms: cordyceps cicadae (Cordyceps cicadae), Isaria sinclairii (Cordyceps sinclairii), Isaria sinclairii (Isaria sinclairii), Paecilomyces cicadae (Paecilomyces cicadae) and Isaria cicadae (Isaria cicadae). Is a cordyceps complex formed after the cordyceps sobolifera fungus infects insects, and has important economic value as a substitute of cordyceps sinensis.
Inhibition of pancreatic lipase activity: pancreatic lipase is the most important enzyme in the digestive system for absorbing fat and plays a key role in the absorption process. The bioactive substance with pancreatic lipase activity can inhibit fat absorption by inhibiting pancreatic lipase activity, so that the bioactive substance can be used for preventing and treating obesity, and has wide application prospect in the field of medicine.
Disclosure of Invention
The object of the present invention is to provide N having an inhibitory activity on pancreatic lipase6A preparation method of-ethyl acetate-3' -acetyl-beta-ribose adenosine.
In order to find a novel natural and highly effective substance having pancreatic lipase inhibitory activity, the inventors of the present invention focused on the prevention and treatment of microorganisms at the university of agriculture, AnhuiActivity research is carried out on metabolites of RECF5833 strain of cicada fungus in laboratory, and a new compound N extracted from culture of cicada fungus is discovered6The-ethyl acetate-3' -acetyl-beta-ribose adenosine has obvious effect of inhibiting the activity of pancreatic lipase.
N with pancreatic lipase activity inhibition function6The-ethyl acetate-3' -ethyl acetate-beta-riboadenosine is prepared by separating Cordyceps cicadae (Cordyceps chanhua) culture, and has molecular formula of C16H21N5O7(ii) a The structural formula is as follows:
said N is6-ethyl acetate-3' -ethyl ester-beta-riboadenosine is yellow oil, and has half Inhibitory Concentration (IC) on pancreatic lipase50) It was 0.267 mg/mL.
N with pancreatic lipase activity inhibition function6The preparation operation steps of the-ethyl acetate-3' -ethyl acetate-beta-ribose adenosine are as follows:
(1) cordyceps sobolifera (Cordyceps chanhua) strain selection
The international bacteria library number of the used cordyceps sobolifera strain is WDCM 1031;
(2) culturing of bacterial strains
The culture method is liquid-solid mixed culture and comprises the following specific steps:
(2.1) seed culture
Inoculating the cordyceps sobolifera strain water solution stock to a Sasa (SDAY) solid plate culture medium, wherein the inoculation amount of each culture dish is 100-300 mu L of cordyceps sobolifera strain water solution, and culturing at the constant temperature of 22-29 ℃ for 8-16 d to obtain a first-level strain;
the formula of the Sasa (SDAY) solid plate culture medium comprises 40g of glucose, 10g of peptone, 10g of yeast extract powder and 20g of agar, and distilled water is added to the medium to reach the constant volume of 1000 mL;
(2.2) Secondary liquid seed culture
The mass of the first-class strain is 0.5-2 cm2Breaking up 100mL, and inoculating the broken powder into a liquid shake flask culture medium for culture;
the liquid shake flask culture medium is: 30-50 g/L of glucose, 8-12 g/L of peptone, 8-12 g/L of yeast extract powder and constant volume of distilled water;
adding a liquid shake flask culture medium with the volume of 30-50% of that of a triangular flask into the triangular flask with the volume of more than or equal to 100mL, wherein each triangular flask is 0.5-2 cm2Inoculating 100mL of the fungus block of the primary strain, placing the fungus block in a constant-temperature oscillation incubator, culturing at the temperature of 22-29 ℃ and the rotating speed of 180-240 rpm for 3-5 days to obtain secondary liquid seeds;
(2.3) solid culture
Inoculating the secondary liquid seeds to a Sasa (SDAY) solid culture medium, wherein the inoculation amount is 150-300 mu L per culture dish, uniformly coating, culturing at the temperature of 22-29 ℃ for 4-8 days at constant temperature, and collecting hyphae to obtain a solid culture;
(3) extraction and refinement of active ingredients in solid cultures
(3.1) extraction of effective Components from solid culture
Performing ultrasonic extraction, membrane filtration or centrifugal separation, and vacuum decompression to remove solvent to obtain effective component extract;
(3.2) preliminary purification of the extract
Primarily purifying the effective component extract by adopting semi-preparative reverse phase high performance liquid chromatography to obtain a yellowish-brown primary purified substance;
(3.3) purification of the preliminary purified product
Refining the yellowish-brown primary purified product by semi-preparative reverse phase high performance liquid chromatography, and collecting the compound corresponding to the first highest peak value under the condition of acetonitrile solution elution to obtain N6Eluting with ethyl acetate-3' -acetyl-beta-riboadenosine, removing solvent under reduced pressure, and drying to obtain yellow oily N6-ethyl acetate-3' -acetyl- β -riboadenosine.
The further preparation operation steps are as follows:
in the step (3.1), the solid culture is dried at the temperature of-50 to 130 ℃, and the weight-volume ratio is 1 g: 0.5-5 mL of ethyl acetate extractant is added into the solid culture; carrying out 40KHz ultrasonic extraction for 20-200 minutes; filtering with 0.20-2.5 μm filter membrane or centrifuging at 4000-15000 rpm to obtain filtrate or centrifugal supernatant; and evaporating the filtrate or the centrifugal supernatant under reduced pressure at the vacuum degree of-0.1 to-0.08 MP and the temperature of 10 to 60 ℃ to remove the extractant, thus obtaining the effective component extract.
In the step (3.2), a semi-preparative liquid chromatogram is utilized, wherein a mobile phase A is ultrapure water, and a mobile phase B is acetonitrile; elution conditions: eluting with 5-60% L of mobile phase B in 0-20 min; the sample injection volume is 20-100 mu L; the column temperature is 20-40 ℃; the flow rate is 2-5 mL/min; the detection wavelength is 260 nm; a chromatographic column: a reversed phase C-18 column; collecting according to peak, and dividing into 8 components, wherein the 6 th component has the required compound; the extractant is evaporated under reduced pressure at the vacuum degree of-0.1 to-0.08 MP and the temperature of 10 to 60 ℃, and the primary yellow-brown purified product is obtained after drying at the temperature of less than or equal to 130 ℃.
In the step (3.3), the chromatographic separation preparation conditions are as follows: the mobile phase A is ultrapure water, and the mobile phase B is acetonitrile; elution conditions: eluting with mobile phase B with 15% to 30% after 0-20 min; the sample injection volume is 20-100 mu L; the column temperature is 20-40 ℃, and the flow rate is 2-5 mL/min; detection wavelength of 260nm, chromatographic column: a reversed phase C-18 column; collecting the compound corresponding to the first highest peak to obtain N6Eluting with ethyl acetate-3' -acetyl-beta-riboadenosine, removing solvent under reduced pressure, and drying to obtain yellow oily N6-ethyl acetate-3' -acetyl- β -riboadenosine.
The analytical study of the present invention is illustrated below:
1. treating Cordyceps cicadae mycelium ethyl acetate extract with liquid chromatography to obtain Cordyceps cicadae extract, which is bioactive purified product N6-ethyl acetate-3' -acetyl- β -riboadenosine.
2. The activity test finds that N6Half inhibitory concentration IC of-acetoxy-3' -acetyl-beta-riboadenosine on pancreatic lipase50) Comprises the following steps: 0.267 mg/ml.
3. Chemical Structure analysis of active Compounds
High resolution LC-MS analysis shows N6-acetic acid ethyl esterEster group-3' -acetyl-beta-ribose adenosine, positive ion 396.1512(M + H), negative ion 394.1434(M-H), and the calculated molecular formula is C16H21N5O7；
NMR data are shown in the following Table
The chemical structure obtained by comprehensive LC-MS analysis and NMR analysis is shown in the following formula
The beneficial technical effects of the invention are embodied in the following aspects:
1. in order to search for a novel natural and efficient substance with pancreatic lipase inhibition activity, the inventor firstly cultures a large number of cordyceps sobolifera strains, then extracts effective components of the culture and measures the activity of the effective components, and finds that the cordyceps sobolifera culture extract has glycosidase inhibition activity. Separating and purifying N by semi-preparative chromatography based on mass culture and extraction6The compound is yellow oily substance, and a structural formula is respectively obtained through one-dimensional and two-dimensional nuclear magnetic resonance and accurate mass spectrum identification. N is a radical of6The-ethyl acetate-3' -acetyl-beta-ribose adenosine has stronger pancreatic lipase inhibition activity, and develops a new application field of cordyceps sobolifera.
2. N prepared by the invention6The-ethyl acetate-3' -acetyl-beta-ribose adenosine has wide application as a pancreatic lipase inhibitor, can be used as a medical drug, and has the effects of reducing blood fat, reducing blood sugar, resisting aging and the like.
3. The invention adopts the microbial fermentation production, is not influenced by environment and resources, is easy to realize industrialization and automation, and is not influenced by environment and natural resources.
4. The process method has the advantages of low production cost, simple and convenient operation, stable process, easy regulation and control and high success rate; the investment of production equipment can be large or small, the production is flexible, and the method is suitable for enterprise investment of various scales.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
N with pancreatic lipase activity inhibition function6The preparation operation steps of the-ethyl acetate-3' -ethyl acetate-beta-ribose adenosine are as follows:
1. cordyceps cicadae strain selection
The international bacteria library number of the used cordyceps sobolifera strain is WDCM 1031.
2. Culturing of bacterial strains
The liquid-solid mixed culture process is adopted, and the specific process operation is as follows:
(2.1) seed culture
Inoculating strain water stock strain of Cordyceps cicadae in Sasa (SDAY) solid plate culture medium with inoculum size of 300 μ L, and culturing at 29 deg.C for 16d to obtain first-class strain;
saxishi (SDAY) solid plate medium composition: 40g of glucose, 10g of peptone, 10g of yeast extract powder and 20g of agar, and adding water to a constant volume of 1000 mL.
(2.2) Secondary liquid seed culture
The mass of the first-class strain is 0.5cm2Dispersing 100mL of the mixture, and inoculating the mixture into a liquid shake flask culture medium for culture;
liquid shake flask culture medium: 50g/L of glucose, 12g/L of peptone, 12g/L of yeast extract powder and constant volume of distilled water;
a500 mL Erlenmeyer flask was charged with 50% Erlenmeyer flask volume of liquid medium, and the inoculum size of each Erlenmeyer flask was 1.25 cm2Inoculating the first-stage strain, placing in a constant-temperature oscillation incubator at 29 deg.C and 240rpm, and culturing for 5d to obtain second-stage liquid seed.
(2.3) solid culture
Inoculating the secondary liquid seeds to a Sasa (SDAY) solid culture medium with the inoculation amount of 300 mu L per culture dish, uniformly coating, putting into a constant-temperature incubator at 29 ℃ for culturing for 8 days, and collecting mycelia to obtain a solid culture.
(3) Extraction and refinement of active ingredients in solid cultures
(3.1) extraction of solid culture
Drying the solid culture at the temperature of 130 ℃, and mixing the solid culture with a solvent according to the weight-volume ratio of 1 g: adding ethyl acetate extractant into 5mL of solid culture, and performing ultrasonic extraction for 200 minutes at 40 KHz; filtering with 2.5 μm filter membrane to obtain filtrate; evaporating the filtrate under reduced pressure at 60 deg.C under vacuum degree of-0.1 MP to remove the extractant to obtain effective component extract.
(3.2) preliminary purification of the extract of the active principle
Utilizing a semi-preparative liquid chromatogram, wherein a mobile phase A is ultrapure water, and a mobile phase B is acetonitrile; elution conditions: eluting with 5-60% of mobile phase B for 0-20 min; the sample introduction volume is 100 mu L, the column temperature is 40 ℃, and the flow rate is 5 mL/min; the detection wavelength is 260 nm; a chromatographic column: a reversed phase C-18 column; collected by peak and divided into 8 fractions with the desired compound in the 6 th fraction. Evaporating under reduced pressure at 60 deg.C under vacuum degree of-0.1 MP to remove extractant, and drying at 130 deg.C to obtain yellowish-brown primary purified product.
(3.3) purification of the preliminary purified product
The chromatographic separation preparation conditions are as follows: the mobile phase A is ultrapure water, and the mobile phase B is acetonitrile; elution conditions: eluting with mobile phase B with 15% to 30% after 0-20 min; the sample introduction volume is 100 mu L, the column temperature is 40 ℃, and the flow rate is 5 mL/min; detection wavelength of 260nm, chromatographic column: a reversed phase C-18 column; collecting the compound corresponding to the first highest peak to obtain N6Eluting with ethyl acetate-3' -ethyl acetate-beta-riboadenosine, removing solvent under reduced pressure, and drying to obtain yellow oily N6-ethyl acetate-3' -ethyl acetate- β -riboadenosine.
The purity is over 90 percent by HPLC-MS analysis. The structure is as follows:
a nucleoside compound purified from an extract of cicada fungus: n is a radical of6-BAcid carbethoxy-3' -carbethoxy-beta-riboadenosine. The activity experiment shows that N6The-ethyl acetate-3' -ethyl ester-beta-ribose adenosine has obvious inhibition effect on the activity of pancreatic lipase and half Inhibition Concentration (IC)50) It was 0.267 mg/mL.
Example 2
N with pancreatic lipase activity inhibition function6The preparation operation steps of the-ethyl acetate-3' -ethyl acetate-beta-ribose adenosine are as follows:
(1) cordyceps cicadae strain selection
The international bacteria library number of the used cordyceps sobolifera strain is WDCM 1031.
(2) Culturing of bacterial strains
The liquid-solid mixed culture process is adopted, and the specific process operation is as follows:
(2.1) seed culture
Inoculating the stock strain of Cordyceps cicadae strain water solution to Sasa (SDAY) solid plate culture medium, inoculating 100 μ l of each plate, and culturing at 22 deg.C for 8d to obtain first-class strain;
(2.2) Secondary liquid seed culture
The mass of the first-class strain is 2cm2Breaking up 100mL, and inoculating the broken powder into a liquid shake flask culture medium for culture;
the liquid shake flask culture medium is prepared by 30g/L glucose, 8g/L peptone, 8g/L yeast extract powder and distilled water to constant volume;
a liquid medium of 30% of the flask volume was added to 100mL flasks, and each flask was inoculated with a solid plate of 0.6cm2Placing the fungus blocks in a constant-temperature oscillation incubator, culturing at 22 ℃ and 180rpm for 3d to obtain secondary liquid seeds;
(2.3) solid culture
Inoculating the secondary liquid seeds to a Sasa (SDAY) solid culture medium, wherein the inoculation amount is 150 mu L per culture dish, uniformly coating, putting into a constant-temperature incubator at 22 ℃ for culturing for 4 days, and collecting mycelia to obtain a solid culture.
(3) Extraction and refinement of active ingredients in solid cultures
(3.1) extraction of solid culture
Drying the solid culture at the temperature of-50 ℃, and mixing the dried solid culture with a solvent according to the weight-volume ratio of 1 g: 0.5mL of ethyl acetate extractant is added into the solid culture, and 40KHz ultrasonic extraction is carried out for 20 minutes; filtering with 0.20 μm filter membrane to obtain filtrate; evaporating the filtrate under reduced pressure at 10 deg.C under vacuum degree of-0.08 MP to remove extractant to obtain effective component extract.
(3.2) preliminary purification of the extract of the active principle
Utilizing a semi-preparative liquid chromatogram, wherein a mobile phase A is ultrapure water, and a mobile phase B is acetonitrile; elution conditions: eluting with 5% to 60% mobile phase B for 0-20 min; the sample injection volume is 20 mu L, the column temperature is 20 ℃, and the flow rate is 2 mL/min; the detection wavelength is 260 nm; a chromatographic column: a reversed phase C-18 column; collected by peak and divided into 8 fractions, with the desired compound in the 6 th fraction. Evaporating under reduced pressure at 10 deg.C under vacuum degree of-0.08 MP to remove extractant, and drying at-50 deg.C to obtain yellowish brown primary purified product.
(3.3) purification of the preliminary purified product
The chromatographic separation preparation conditions are as follows: the mobile phase A is ultrapure water, and the mobile phase B is acetonitrile; elution conditions: eluting with mobile phase B15% to 30% for 0-20 min; the sample injection volume is 20 mu L, the column temperature is 20 ℃, and the flow rate is 2 mL/min; detection wavelength of 260nm, chromatographic column: a reversed phase C-18 column; collecting the compound corresponding to the first highest peak to obtain N6Eluting with ethyl acetate-3' -ethyl acetate-beta-riboadenosine, removing solvent under reduced pressure, and drying to obtain yellow oily N6-ethyl acetate-3' -ethyl acetate- β -riboadenosine.
The purity is over 90 percent by HPLC-MS analysis. The structure is as follows:
a nucleoside compound N purified from Cordyceps cicadae extract6Activity experiments show that the compound has obvious inhibition effect on pancreatic lipase activity and half Inhibition Concentration (IC)50) It was 0.267 mg/mL.
Example 3
N with pancreatic lipase activity inhibition function6The preparation operation steps of the-ethyl acetate-3' -ethyl acetate-beta-ribose adenosine are as follows:
(1) cordyceps cicadae strain selection
The international bacteria library number of the used cordyceps sobolifera strain is WDCM 1031.
(2) Culturing of bacterial strains
The liquid-solid mixed culture process is adopted, and the specific process operation is as follows:
(2.1) seed culture
Inoculating Cordyceps cicadae strain water solution stock strain to Sasa (SDAY) solid plate culture medium, inoculating 200 μ L of each plate, and culturing at constant temperature of 25 deg.C for 12d to obtain first-class strain;
(2.2) Secondary liquid seed culture
The mass of the first-class strain is 1cm2Breaking up 100mL, and inoculating the broken powder into a liquid shake flask culture medium for culture;
the liquid shake flask culture medium is prepared by 40g/L glucose, 10g/L peptone, 10g/L yeast extract powder and distilled water to constant volume;
a250 mL Erlenmeyer flask was filled with 40% Erlenmeyer flask volume of liquid medium, and each Erlenmeyer flask was inoculated with a 1cm piece of solid flat plate fungus2Placing the seeds in a constant-temperature oscillation incubator, and culturing for 4d at the temperature of 25 ℃ and the rotating speed of 200rpm to obtain secondary liquid seeds;
(2.3) solid culture
Inoculating the secondary liquid seeds to a Sasa (SDAY) solid culture medium, wherein the inoculation amount is 225 μ L per culture dish, uniformly coating, placing in a constant-temperature incubator at 25 ℃ for culturing for 6 days, and collecting mycelia to obtain a solid culture.
(3) Extraction and refinement of active ingredients in solid cultures
(3.1) extraction of solid culture
Drying the solid culture at the temperature of 60 ℃, and mixing the solid culture with a solvent according to the weight-volume ratio of 1 g: adding ethyl acetate extractant into 3mL of solid culture, and performing ultrasonic extraction for 120 minutes by using 40 KHz; centrifuging at 15000 r/min to obtain supernatant; centrifuging the supernatant, and evaporating under reduced pressure at 40 deg.C under vacuum degree of-0.09 MP to remove the extractant to obtain effective component extract.
(3.2) preliminary purification of the extract of the active principle
Utilizing a semi-preparative liquid chromatogram, wherein a mobile phase A is ultrapure water, and a mobile phase B is acetonitrile; elution conditions: eluting with 5% to 60% mobile phase B for 0-20 min; the sample introduction volume is 60 mu L, the column temperature is 30 ℃, and the flow rate is 3.5 mL/min; the detection wavelength is 260 nm; a chromatographic column: a reversed phase C-18 column; collected by peak and divided into 8 fractions, the compound in fraction 6. Evaporating under reduced pressure at 40 deg.C under vacuum degree of-0.09 MP to remove extractant, and drying at 60 deg.C to obtain yellowish brown primary purified product.
(3.3) purification of the preliminary purified product
The chromatographic separation preparation conditions are as follows: the mobile phase A is ultrapure water, and the mobile phase B is acetonitrile; elution conditions: eluting with mobile phase B15% to 30% for 0-20 min; the sample injection volume is 20 mu L, the column temperature is 30 ℃, and the flow rate is 3.5 mL/min; detection wavelength of 260nm, chromatographic column: a reversed phase C-18 column; collecting the compound corresponding to the first highest peak to obtain N6Eluting with ethyl acetate-3' -ethyl acetate-beta-riboadenosine, removing solvent under reduced pressure, and drying to obtain yellow oily N6-ethyl acetate-3' -ethyl acetate- β -riboadenosine.
The purity is over 90% by HPLC-MS analysis, and the structure is as follows:
a nucleoside compound N purified from Cordyceps cicadae extract6Activity experiments show that the compound has obvious inhibition effect on pancreatic lipase activity and half Inhibition Concentration (IC)50) It was 0.267 mg/mL.
Example 4
N with pancreatic lipase activity inhibition function6The preparation operation steps of the-ethyl acetate-3' -ethyl acetate-beta-ribose adenosine are as follows:
(1) cordyceps cicadae strain selection
The international bacteria library number of the used cordyceps sobolifera strain is WDCM 1031.
(2) Culturing of bacterial strains
The liquid-solid mixed culture process is adopted, and the specific process operation is as follows:
(2.1) seed culture
Inoculating Cordyceps cicadae strain water solution stock strain to Sasa (SDAY) solid plate culture medium, inoculating 200 μ L of each plate, and culturing at constant temperature of 25 deg.C for 12d to obtain first-class strain;
(2.2) Secondary liquid seed culture
The mass of the first-class strain is 1cm2Breaking up by 100mL, and inoculating into a liquid culture shake flask culture medium for culture;
the liquid shake flask culture medium is prepared by 40g/L glucose, 10g/L peptone, 10g/L yeast extract powder and distilled water to constant volume;
a250 mL Erlenmeyer flask was filled with 40% Erlenmeyer flask volume of liquid medium, and each Erlenmeyer flask was inoculated with a 1cm piece of solid flat plate fungus2Placing the seeds in a constant-temperature oscillation incubator, and culturing for 4d at the temperature of 25 ℃ and the rotating speed of 200rpm to obtain secondary liquid seeds;
(2.3) solid culture
Inoculating the secondary liquid seeds to a Sasa (SDAY) solid culture medium, wherein the inoculation amount is 225 μ L per culture dish, uniformly coating, placing in a constant-temperature incubator at 25 ℃ for culturing for 6 days, and collecting mycelia to obtain a solid culture.
(3) Extraction and refinement of active ingredients in solid cultures
(3.1) extraction of solid culture
Drying the solid culture at the temperature of 60 ℃, and mixing the solid culture with a solvent according to the weight-volume ratio of 1 g: adding ethyl acetate extractant into 3mL of solid culture, and performing ultrasonic extraction for 120 minutes by using 40 KHz; centrifuging at 4000 rpm to obtain a centrifugal supernatant; centrifuging the supernatant, and evaporating under reduced pressure at 40 deg.C under vacuum degree of-0.09 MP to remove the extractant to obtain effective component extract.
(3.2) preliminary purification of the extract of the active principle
Utilizing a semi-preparative liquid chromatogram, wherein a mobile phase A is ultrapure water, and a mobile phase B is acetonitrile; elution conditions: eluting with 5% to 60% mobile phase B for 0-20 min; the sample introduction volume is 60 mu L, the column temperature is 30 ℃, and the flow rate is 3 mL/min; the detection wavelength is 260 nm; a chromatographic column: a reversed phase C-18 column; collected by peak and divided into 8 fractions, the compound in fraction 6. Evaporating under reduced pressure at 40 deg.C under vacuum degree of-0.09 MP to remove extractant, and drying at 100 deg.C to obtain yellowish brown primary purified product.
(3.3) purification of the preliminary purified product
The chromatographic separation preparation conditions are as follows: the mobile phase A is ultrapure water, and the mobile phase B is acetonitrile; elution conditions: eluting with mobile phase B15% to 30% for 0-20 min; the sample injection volume is 20 mu L, the column temperature is 30 ℃, and the flow rate is 3 mL/min; detection wavelength of 260nm, chromatographic column: a reversed phase C-18 column; collecting the compound corresponding to the first highest peak to obtain N6Eluting with ethyl acetate-3' -ethyl acetate-beta-riboadenosine, removing solvent under reduced pressure, and drying to obtain yellow oily N6-ethyl acetate-3' -ethyl acetate- β -riboadenosine.
The purity is over 90 percent by HPLC-MS analysis, and the structural formula is as follows:
a nucleoside compound N purified from Cordyceps cicadae extract6Activity experiments show that the compound has obvious inhibition effect on pancreatic lipase activity and half Inhibition Concentration (IC)50) It was 0.267 mg/mL.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (5)
1. N with pancreatic lipase activity inhibition function6-ethyl acetate-3' -ethyl acetate- β -riboadenosine, characterized by: is isolated from culture of Cordyceps cicadae (Cordyceps chanhua)Prepared by the method of preparing the compound with the molecular formula of C16H21N5O7(ii) a The structural formula is as follows:
said N is6-ethyl acetate-3' -ethyl ester-beta-riboadenosine is yellow oil, and has half Inhibitory Concentration (IC) on pancreatic lipase50) It was 0.267 mg/mL.
2. N with pancreatic lipase inhibiting activity according to claim 16The preparation method of the-ethyl acetate-3' -ethyl acetate-beta-ribose adenosine is characterized by comprising the following specific operation steps:
(1) cordyceps sobolifera (Cordyceps chanhua) strain selection
The international bacteria library number of the used cordyceps sobolifera strain is WDCM 1031;
(2) culturing of bacterial strains
The culture method is liquid-solid mixed culture and comprises the following specific steps:
(2.1) seed culture
Inoculating the cordyceps sobolifera strain water solution protospecies to a Sasa solid plate culture medium, wherein the inoculation amount of each culture dish is 100-300 mu L, and culturing at the constant temperature of 22-29 ℃ for 8-16 d to obtain a first-level strain;
the formula of the Sa's solid plate culture medium comprises 40g of glucose, 10g of peptone, 10g of yeast extract powder and 20g of agar, and distilled water is added to the medium to reach the constant volume of 1000 mL;
(2.2) Secondary liquid seed culture
The mass of the first-class strain is 0.5-2 cm2Breaking up 100mL, and inoculating the broken powder into a liquid shake flask culture medium for culture;
the liquid shake flask culture medium is: 30-50 g/L of glucose, 8-12 g/L of peptone, 8-12 g/L of yeast extract powder and constant volume of distilled water;
adding a liquid shake flask culture medium with the volume of 30-50% of that of a triangular flask into the triangular flask with the volume of more than or equal to 100mL, wherein each triangular flask is 0.5-2 cm2100mL ofInoculating the fungus block of the primary strain, placing the fungus block in a constant-temperature oscillation incubator, culturing at the temperature of 22-29 ℃ and the rotating speed of 180-240 rpm for 3-5 days to obtain secondary liquid seeds;
(2.3) solid culture
Inoculating the secondary liquid seeds onto a Sasa solid culture medium, wherein the inoculation amount is 150-300 mu L per culture dish, uniformly coating, culturing at the constant temperature of 22-29 ℃ for 4-8 days, and collecting mycelia to obtain a solid culture;
(3) extraction and refinement of active ingredients in solid cultures
(3.1) extraction of effective Components from solid culture
Performing ultrasonic extraction, membrane filtration or centrifugal separation, and vacuum decompression to remove solvent to obtain effective component extract;
(3.2) preliminary purification of the extract
Primarily purifying the effective component extract by adopting semi-preparative reverse phase high performance liquid chromatography to obtain a yellowish-brown primary purified substance;
(3.3) purification of the preliminary purified product
Refining the yellowish-brown primary purified product by semi-preparative reverse phase high performance liquid chromatography, and collecting the compound corresponding to the first highest peak value under the condition of acetonitrile solution elution to obtain N6Eluting with ethyl acetate-3' -acetyl-beta-riboadenosine, removing solvent under reduced pressure, and drying to obtain yellow oily N6-ethyl acetate-3' -acetyl- β -riboadenosine.
3. The method of claim 2, wherein: in the step (3.1), the solid culture is dried at the temperature of-50 to 130 ℃, and the weight-volume ratio is 1 g: 0.5-5 mL of ethyl acetate extractant is added into the solid culture; carrying out 40KHz ultrasonic extraction for 20-200 minutes; filtering with 0.20-2.5 μm filter membrane or centrifuging at 4000-15000 rpm to obtain filtrate or centrifugal supernatant; and evaporating the filtrate or the centrifugal supernatant under reduced pressure at the vacuum degree of-0.1 to-0.08 MP and the temperature of 10 to 60 ℃ to remove the extractant, thus obtaining the effective component extract.
4. The method of claim 2, wherein: in the step (3.2), a semi-preparative liquid chromatogram is utilized, wherein a mobile phase A is ultrapure water, and a mobile phase B is acetonitrile; elution conditions: eluting with 5-60% L of mobile phase B in 0-20 min; the sample injection volume is 20-100 mu L; the column temperature is 20-40 ℃; the flow rate is 2-5 mL/min; the detection wavelength is 260 nm; a chromatographic column: a reversed phase C-18 column; collecting according to peak, and dividing into 8 components, wherein the 6 th component has the required compound; and (3) evaporating the extractant under reduced pressure at the vacuum degree of-0.1 to-0.08 MP and the temperature of 10 to 60 ℃, and drying at the temperature of less than or equal to 130 ℃ to obtain a yellowish-brown primary purified product.
5. The method of claim 2, wherein: in the step (3.3), the chromatographic separation preparation conditions are as follows: the mobile phase A is ultrapure water, and the mobile phase B is acetonitrile; elution conditions: eluting with mobile phase B with 15% to 30% after 0-20 min; the sample injection volume is 20-100 mu L; the column temperature is 20-40 ℃, and the flow rate is 2-5 mL/min; detection wavelength of 260nm, chromatographic column: a reversed phase C-18 column; collecting the compound corresponding to the first highest peak to obtain N6Eluting with ethyl acetate-3' -acetyl-beta-riboadenosine, removing solvent under reduced pressure, and drying to obtain yellow oily N6-ethyl acetate-3' -acetyl- β -riboadenosine.
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| SEON BEOM KIM等: "Effect of Cordyceps militaris extract and active constituents on metabolic parameters of obesity induced by high-fat diet in C58BL/6J mice", 《JOURNAL OF ETHNOPHARMACOLOGY》, vol. 151, pages 512 - 513 * |
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