CN114276395A - N having pancreatic lipase inhibitory activity6-hydroxyethyl-5' -acetyl-beta-ribose adenosine and preparation method thereof - Google Patents
N having pancreatic lipase inhibitory activity6-hydroxyethyl-5' -acetyl-beta-ribose adenosine and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to N having an inhibitory activity on pancreatic lipase6-hydroxyethyl-5' -acetyl-beta-ribose adenosine and a preparation method thereof, belonging to the technical field of extraction and preparation of biological products. N is a radical of6-hydroxyethyl-5' -acetyl-beta-riboadenosine is a white powdery substance, and is processed by one-dimensional and two-dimensionalRespectively obtaining N by nuclear magnetic resonance and accurate mass spectrum identification6-hydroxyethyl-5' -acetyl-beta-riboadenosine structural formula. N of the invention6The-hydroxyethyl-5' -acetyl-beta-ribose adenosine is obtained by culturing cicada fungus and then extracting and separating. N of the invention6The-hydroxyethyl-5' -acetyl-beta-ribose adenosine 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 relates to N with pancreatic lipase inhibitory activity6Extraction, purification, structural identification and activity determination of-hydroxyethyl-5' -acetyl-beta-riboadenosine.
Background
N6-hydroxyethyl-5' -acetyl-beta-riboadenosine is a novel bioactive substance with pancreatic lipase inhibitory activity isolated from a culture of cicada fungus (cordyces chanhua).
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 having pancreatic lipase activity can inhibit fat absorption by inhibiting pancreatic lipase activity, and thus can be used for preventing and treating obesity, etc., and the cicada fungus is a medicinal fungus having great effects in daily health care, blood fat lowering and blood sugar lowering. Has wide application prospect in the field of medicine.
Disclosure of Invention
In order to search for a novel natural and efficient substance with pancreatic lipase inhibitory activity, the inventor conducts activity research on a metabolite of the RECF5833 strain of cordyceps sobolifera in key laboratory through microbial control of university of agriculture in Anhui, and finds out a novel compound N extracted from cordyceps sobolifera culture6The-hydroxyethyl-5' -acetyl-beta-ribose adenosine has obvious effect of inhibiting the activity of pancreatic lipase.
N with pancreatic lipase activity inhibition function6-hydroxyethyl-5' -acetyl-beta-riboadenosine is isolated from a culture of cicada fungus (Cordyceps chanhua) and has the following structural formula:
said N is6-hydroxyethyl-5' -acetyl-beta-riboadenosine is in the form of a white powder with a semi-inhibitory concentration IC50 for pancreatic lipase of 200 μ g/mL.
The N has the function of inhibiting the activity of pancreatic lipase6The preparation operation steps of the-hydroxyethyl-5' -acetyl-beta-ribose adenosine are as follows:
(1) preparation of solid cultures
Taking a cordyceps sobolifera strain of an international bacteria library number WDCM1031, and carrying out liquid culture and solid culture on the strain, wherein the specific operation procedures are as follows:
(1.1) first-order seed culture
Inoculating the cordyceps sobolifera strain liquid stock to a Sasa (SDAY) solid plate culture medium, inoculating 100-300 mu L of the cordyceps sobolifera strain liquid stock to each culture dish, and culturing the cordyceps sobolifera strain liquid stock for 8-16 days at a constant temperature of 22-29 ℃ to obtain a first-class 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 water is added to the medium to reach the constant volume of 1000 mL;
(1.2) Secondary liquid seed culture
The mass of the first-class strain is 0.5-2 cm2Breaking up by 100mL, and inoculating into a liquid shake flask culture medium;
liquid shake flask culture medium: 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 culture medium with the volume of 30-50% of that of a triangular flask into a triangular flask with the volume of more than or equal to 100mL, inoculating the broken solid plate bacterial block on each triangular flask, and carrying out constant-temperature shaking culture at the temperature of 22-29 ℃ and the rotation speed of 180-240 rpm for 3-5 days to obtain secondary liquid seeds;
(1.3) 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 for 4-8 days at a constant temperature of 22-29 ℃, collecting mycelia, and drying at a temperature of-50-130 ℃ to obtain a dried solid culture;
(2) extraction and refinement of active ingredients in culture
The specific operation is as follows:
(2.1) 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;
(2.2) preliminary purification of the extract
Performing preliminary purification of the extract of the effective component by semi-preparative reverse phase high performance liquid chromatography to obtain the fourth component containing the desired compound; evaporating the extractant at a vacuum degree of-0.1 to-0.08 MP and a temperature of 10 to 60 ℃ under reduced pressure, and drying at a temperature lower than 150 ℃ to obtain a white primary purified product;
(2.3) purification
Refining the yellowish-brown primary purified substance 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-hydroxyethyl-5' -acetyl-beta-riboadenosine, removing solvent under reduced pressure, and drying to obtain white powder of N6-hydroxyethyl-5' -acetyl- β -riboadenosine.
The preparation operation steps are further defined as follows:
in the step (2.1), the solid culture is added into a culture medium according to the weight-volume ratio of 1 g: adding 0.5-5 mL of ethyl acetate, and performing 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 to remove ethyl acetate under reduced pressure at the vacuum degree of-0.1 to-0.08 MP and the temperature of 10 to 60 ℃ to obtain the active ingredient extract.
In the step (2.2), 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% to 60% mobile phase B for 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 peaks to obtain eight components, wherein the fourth 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 white primary purified product.
In the step (2.3), 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 15% mobile phase B for 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; 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-hydroxyethyl-5' -acetyl-beta-riboadenosine, removing solvent under reduced pressure, and drying to obtain white powder of N6-hydroxyethyl-5' -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 with bioactivity, N6-hydroxyethyl-5' -acetyl- β -riboadenosine.
2. The activity test finds that N6Half maximal Inhibitory Concentration (IC) of-hydroxyethyl-5' -acetyl-beta-riboadenosine on pancreatic lipase50) Comprises the following steps: 200. mu.g/mL.
3. Chemical Structure analysis of active Compounds
High resolution LC-MS analysis shows N6-hydroxyethyl-5' -acetyl- β -riboadenosine, positive 354.1472(M + H), negative 352.1396(M-H), calculated as C14H19N5O6；
Nmr analysis data are shown in the table below.
The chemical structure obtained by comprehensive liquid chromatography-mass spectrometry analysis and nuclear magnetic resonance analysis is shown in the following figure:
the beneficial technical effects of the invention are shown in the following:
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 and measures the activity of the effective components of the culture, and finds that a cordyceps sobolifera culture extract has pancreatic lipase inhibition activity. Separating and purifying N by semi-preparative chromatography based on mass culture and extraction6-hydroxyethyl-5' -acetyl-beta-riboadenosine, which is white powder and has a structural formula respectively obtained by one-dimensional and two-dimensional nuclear magnetic resonance and accurate mass spectrometric identification. N is a radical of6The-hydroxyethyl-5' -acetyl-beta-ribose adenosine has stronger pancreatic lipase inhibition activity, and develops a new application field of cordyceps sobolifera.
2. The pancreatic lipase inhibitor prepared by the invention has wide application, 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 product produced by the process method has low cost, simple and convenient process, stable process, easy regulation and control and high success rate.
5. The invention has the advantages of changeable investment of production equipment, flexible production and suitability for investment of enterprises of various scales.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
N having pancreatic lipase inhibitory activity6The preparation operation steps of the-hydroxyethyl-5' -acetyl-beta-ribose adenosine are as follows:
(1) preparation of solid cultures
Taking a cordyceps sobolifera strain of an international bacteria library number WDCM1031, and carrying out liquid culture and solid culture on the strain, wherein the specific operation procedures are as follows:
(1.1) first-order seed culture
Inoculating Cordyceps cicadae strain liquid stock to Sasa (SDAY) solid plate culture medium, inoculating 300 μ L of each plate, and culturing at 29 deg.C for 16d to obtain first-class strain;
(1.2) Secondary liquid seed culture
The first-class strain block is divided into 2cm2The solution is scattered by 100mL and inoculated 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 filled with liquid medium in a volume of 50% Erlenmeyer flask, and each Erlenmeyer flask was inoculated with 5cm of a pellet on a solid plate2After inoculation, placing the mixture in a constant-temperature oscillation incubator, and culturing for 5d at the temperature of 29 ℃ and the rotating speed of 240rpm to obtain a secondary strain;
(1.3) 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, collecting mycelia, and drying at 130 ℃ to obtain a dry solid culture.
(2) Extraction and refinement of active ingredients in culture
The specific operation is as follows:
(2.1) extraction of effective Components from solid culture
In the solid culture, the weight volume ratio of 1 g: adding ethyl acetate into 5mL of the mixture for extraction, and performing ultrasonic extraction on the mixture 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.
(2.2) preliminary purification of the extract
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 injection volume is 100 mu L; the column temperature is 40 ℃; the flow rate is 5 mL/min; the detection wavelength is 260 nm; a chromatographic column: a reversed phase C-18 column; collect by peak to give 8 fractions with the desired compound in fraction 4. 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 white primary purified product.
(2.3) 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 15% mobile phase B for 0-20 min; the sample injection volume is 100 mu L; the column temperature is 40 ℃; 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-hydroxyethyl-5' -acetyl-beta-riboadenosine, removing solvent under reduced pressure, and drying to obtain white powder of N6-hydroxyethyl-5' -acetyl- β -riboadenosine. The purity is over 90% by HPLC-MS analysis, and the structure is as follows:
a nucleoside compound purified from an extract of cicada fungus: n is a radical of6The activity experiment shows that the compound has obvious inhibition effect on the activity of pancreatic lipaseHalf maximal Inhibitory Concentration (IC)50) 200. mu.g/mL.
Example 2
N having pancreatic lipase inhibitory activity6The preparation operation steps of the-hydroxyethyl-5' -acetyl-beta-ribose adenosine are as follows:
(1) preparation of solid cultures
Taking a cordyceps sobolifera strain of an international bacteria library number WDCM1031, and carrying out liquid culture and solid culture on the strain, wherein the specific operation procedures are as follows:
(1.1) first-order seed culture
Inoculating Cordyceps cicadae strain liquid stock to Sasa (SDAY) solid plate culture medium, inoculating 100 μ L each plate, and culturing at constant temperature of 22 deg.C for 8d to obtain first-class strain.
(1.2) Secondary liquid seed culture
The mass of the first-class strain is 0.5cm2Breaking up 100mL, and inoculating the broken powder into a liquid shake flask culture medium for culture;
liquid shake flask culture medium: 30g/L of glucose, 8g/L of peptone, 8g/L of yeast extract powder and constant volume of distilled water;
a liquid medium of 30% of the flask volume was added to 100mL flasks, and each flask was inoculated with a solid plate of 0.15cm2And (3) placing the bacterium blocks in a constant-temperature shaking incubator, and culturing for 3d at the temperature of 22 ℃ and the rotating speed of 180rpm to obtain secondary liquid seeds.
(1.3) solid culture
Inoculating the secondary liquid seeds to a Sasa (SDAY) solid culture medium with the inoculation amount of 150 μ L per culture dish, uniformly coating, culturing in a constant-temperature incubator at 22 deg.C for 4 days, collecting mycelia, and drying at-50 deg.C to obtain a dry solid culture;
(2) extraction and refinement of active ingredients in culture
The specific operation is as follows:
(2.1) extraction of effective Components from solid culture
In the solid culture, the weight volume ratio of 1 g: adding 0.5mL of ethyl acetate extractant, and performing 40KHz ultrasonic extraction 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.
(2.2) 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 injection volume is 20 mu L; the column temperature is 20 ℃; 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, the compound in fraction 4. 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 white primary purified product.
(2.3) 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 15% mobile phase B for 0-20 min; the injection volume is 20 mu L; the column temperature is 20 ℃; 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-hydroxyethyl-5' -acetyl-beta-riboadenosine, removing solvent under reduced pressure, and drying to obtain white powder of N6-hydroxyethyl-5' -acetyl- β -riboadenosine. The purity is over 90% by HPLC-MS analysis, and the structure is as follows:
a nucleoside compound purified from an extract of cicada fungus: n is a radical of6The activity experiment shows that the compound has obvious inhibition effect on the activity of pancreatic lipase and half Inhibition Concentration (IC)50) 200. mu.g/mL.
Example 3
N having pancreatic lipase inhibitory activity6The preparation operation steps of the-hydroxyethyl-5' -acetyl-beta-ribose adenosine are as follows:
(1) preparation of solid cultures
Taking a cordyceps sobolifera strain of an international bacteria library number WDCM1031, and carrying out liquid culture and solid culture on the strain, wherein the specific operation procedures are as follows:
(1.1) first-order seed culture
Inoculating Cordyceps cicadae strain liquid stock to Sasa (SDAY) solid plate culture medium, inoculating 200 μ L each plate, and culturing at constant temperature of 25 deg.C for 12d to obtain first-class strain.
(1.2) 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;
liquid shake flask culture medium: 40g/L of glucose, 10g/L of peptone, 10g/L of yeast extract powder and constant volume of distilled water;
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 fungus2And after inoculation, placing 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.
(1.3) solid culture
Inoculating the secondary liquid seeds to a Sasa (SDAY) solid culture medium with an inoculum size of 225 μ L per culture dish, uniformly coating, culturing in a constant temperature incubator at 25 deg.C for 6 days, collecting mycelia, and drying at 70 deg.C to obtain dry solid culture.
(2) Extraction and refinement of active ingredients in culture
The specific operation is as follows:
(2.1) extraction of effective Components from solid culture
In the solid culture, the weight volume ratio of 1 g: adding ethyl acetate extractant into 3mL of the mixture, and performing ultrasonic extraction on the mixture for 120 minutes at 40 KHz; centrifuging at 15000 r/min to obtain supernatant; centrifuging the supernatant, and evaporating to remove ethyl acetate under reduced pressure at 40 deg.C under vacuum degree of-0.09 MP to obtain effective component extract.
(2.2) 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 60 mu L; the column temperature is 30 ℃; 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 4. Evaporating under reduced pressure at 40 deg.C under vacuum degree of-0.09 MP to remove extractant, and drying at-30 deg.C to obtain white primary purified product.
(2.3) 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 15% mobile phase B for 0-20 min; the sample injection volume is 60 mu L; the column temperature is 30 ℃; 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-hydroxyethyl-5' -acetyl-beta-riboadenosine, removing solvent under reduced pressure, and drying to obtain white powder of N6-hydroxyethyl-5' -acetyl- β -riboadenosine. The purity is over 90% by HPLC-MS analysis, and the structure is as follows:
a nucleoside compound purified from an extract of cicada fungus: n is a radical of6The activity experiment shows that the compound has obvious inhibition effect on the activity of pancreatic lipase and half Inhibition Concentration (IC)50) 200. mu.g/mL.
Example 4
N having pancreatic lipase inhibitory activity6The preparation operation steps of the-hydroxyethyl-5' -acetyl-beta-ribose adenosine are as follows:
(1) preparation of solid cultures
Taking a cordyceps sobolifera strain of an international bacteria library number WDCM1031, and carrying out liquid culture and solid culture on the strain, wherein the specific operation procedures are as follows:
(1.1) first-order seed culture
Inoculating Cordyceps cicadae strain liquid stock to Sasa (SDAY) solid plate culture medium, inoculating 200 μ L each plate, and culturing at constant temperature of 25 deg.C for 12d to obtain first-class strain.
(1.2) 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;
liquid shake flask culture medium: 40g/L of glucose, 10g/L of peptone, 10g/L of yeast extract powder and constant volume of distilled water;
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 fungus2And placing the seeds in a constant-temperature oscillation incubator, culturing for 4d at the temperature of 25 ℃ and the rotating speed of 200rpm to obtain secondary liquid seeds.
(1.3) solid culture
Inoculating the secondary liquid seeds to a Sasa (SDAY) solid culture medium with an inoculum size of 225 μ L per culture dish, uniformly coating, culturing in a constant temperature incubator at 25 deg.C for 6 days, collecting mycelia, and drying at 60 deg.C to obtain dry solid culture.
(2) Extraction and refinement of active ingredients in culture
The specific operation is as follows:
(2.1) extraction of effective Components from solid culture
In the solid culture, the weight volume ratio of 1 g: adding ethyl acetate extractant into 3mL of the mixture, and performing ultrasonic extraction on the mixture for 120 minutes at 40 KHz; centrifuging at 4000 rpm to obtain a centrifugal supernatant; centrifuging the supernatant, and evaporating to remove ethyl acetate under reduced pressure at 40 deg.C under vacuum degree of-0.09 MP to obtain effective component extract.
(2.2) 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 60 mu L; the column temperature is 30 ℃; the flow rate is 3.0 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 desired compound in the 4 th fraction. Evaporating under reduced pressure at 40 deg.C under vacuum degree of-0.09 MP to remove extractant, and drying at-30 deg.C to obtain white primary purified product.
(2.3) 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 15% mobile phase B for 0-20 min; the sample injection volume is 60 mu L; the column temperature is 30 ℃; the flow rate is 3.0 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-hydroxyethyl-5' -acetyl-beta-riboadenosine, removing solvent under reduced pressure, and drying to obtain white powder of N6-hydroxyethyl-5' -acetyl- β -riboadenosine. The purity is over 90% by HPLC-MS analysis, and the structure is as follows:
a nucleoside compound purified from an extract of cicada fungus: n is a radical of6The activity experiment shows that the compound has obvious inhibition effect on the activity of pancreatic lipase and half Inhibition Concentration (IC)50) 200. mu.g/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-hydroxyethyl-5' -acetyl- β -riboadenosine, characterized by: is prepared by separating Cordyceps sobolifera (Cordyceps chanhua) culture, and has the following structural formula:
said N is6-hydroxyethyl-5' -acetyl-beta-riboadenosine is in the form of a white powder with a semi-inhibitory concentration IC50 for pancreatic lipase of 200 μ g/mL.
2. N according to claim 16The preparation method of the (E) -hydroxyethyl-5' -acetyl-beta-ribose adenosine is characterized by comprising the following operation steps:
(1) preparation of solid cultures
Taking a cordyceps sobolifera strain of an international bacteria library number WDCM1031, and carrying out liquid culture and solid culture on the strain, wherein the specific operation procedures are as follows:
(1.1) first-order seed culture
Inoculating the cordyceps sobolifera strain liquid stock to a Sasa (SDAY) solid plate culture medium, inoculating 100-300 mu L of the cordyceps sobolifera strain liquid stock to each culture dish, and culturing the cordyceps sobolifera strain liquid stock for 8-16 days at a constant temperature of 22-29 ℃ to obtain a first-class 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 water is added to the medium to reach the constant volume of 1000 mL;
(1.2) Secondary liquid seed culture
The mass of the first-class strain is 0.5-2 cm2Breaking up by 100mL, and inoculating into a liquid shake flask culture medium;
liquid shake flask culture medium: 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 culture medium with the volume of 30-50% of that of a triangular flask into a triangular flask with the volume of more than or equal to 100mL, inoculating the broken solid plate bacterial block on each triangular flask, and carrying out constant-temperature shaking culture at the temperature of 22-29 ℃ and the rotation speed of 180-240 rpm for 3-5 days to obtain secondary liquid seeds;
(1.3) 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 for 4-8 days at a constant temperature of 22-29 ℃, collecting mycelia, and drying at a temperature of-50-130 ℃ to obtain a dried solid culture;
(2) extraction and refinement of active ingredients in culture
The specific operation is as follows:
(2.1) 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;
(2.2) preliminary purification of the extract
Performing preliminary purification of the extract of the effective component by semi-preparative reverse phase high performance liquid chromatography to obtain the fourth component containing the desired compound; evaporating the extractant at a vacuum degree of-0.1 to-0.08 MP and a temperature of 10 to 60 ℃ under reduced pressure, and drying at a temperature lower than 150 ℃ to obtain a white primary purified product;
(2.3) purification
Refining the yellowish-brown primary purified substance 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-hydroxyethyl-5' -acetyl-beta-riboadenosine, removing solvent under reduced pressure, and drying to obtain white powder of N6-hydroxyethyl-5' -acetyl- β -riboadenosine.
3. The method of claim 2, wherein: in the step (2.1), the solid culture is added into a culture medium according to the weight-volume ratio of 1 g: adding 0.5-5 mL of ethyl acetate, and performing 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 to remove ethyl acetate under reduced pressure at the vacuum degree of-0.1 to-0.08 MP and the temperature of 10 to 60 ℃ to obtain the active ingredient extract.
4. The method of claim 2, wherein: in the step (2.2), 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% to 60% mobile phase B for 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 peaks to obtain eight components, wherein the fourth 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 white primary purified product.
5. The method of claim 2, wherein: in the step (2.3), 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 15% mobile phase B for 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; 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-hydroxyethyl-5' -acetyl-beta-riboadenosine, removing solvent under reduced pressure, and drying to obtain white powder of N6-hydroxyethyl-5' -acetyl- β -riboadenosine.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114057806A (en) * | 2021-11-12 | 2022-02-18 | 贵州大学 | Method for separating and purifying HEA in paecilomyces cicadae 5704s mycelium |
| NL2033899B1 (en) * | 2022-10-21 | 2024-05-08 | Univ Anhui Agricultural | Four nucleosides of chanhua having activity of promoting proliferation of macrophages and renal cells and preparation method thereof |
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