CN114315936A - N6-ethyl acetate-5' -acetyl-beta-ribose adenosine with pancreatic lipase activity inhibition function and preparation method thereof - Google Patents
N6-ethyl acetate-5' -acetyl-beta-ribose adenosine with pancreatic lipase activity inhibition function 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-5' -ethyl ester-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-5' -ethyl ester-beta-ribose adenosine is yellow oily substance which is subjected to one-dimensional neutralizationRespectively obtaining N by two-dimensional nuclear magnetic resonance and accurate mass spectrum identification6-ethyl acetate-5' -ethyl ester-beta-ribose adenosine structural formula. The required compound is obtained by culturing the cordyceps sobolifera fungus and then extracting and separating. N prepared by the invention6The-ethyl acetate-5' -ethyl ester-beta-ribose adenosine can be used for preparing pancreatic lipase inhibitors, has potential effects on treating obesity and also has potential effects 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 function6-ethyl acetate-5' -acetyl-beta-ribose adenosine and its extraction, purification, structure identification and activity determination.
Background
N6-ethyl acetate-5’Acetyl-beta-riboadenosine is a novel bioactive substance having pancrelipase inhibitory activity isolated from a culture of a fungus cicada fungus (Cordyceps chanhua).
Cordyceps cicadae (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
To look for a new dayHowever, the inventor conducts activity research through a cordyceps sobolifera RECF5833 strain metabolite to find a novel compound N extracted from a cordyceps sobolifera fungus culture6-ethyl acetate-5’The acetyl-beta-ribose adenosine has obvious function of inhibiting the activity of pancreatic lipase.
N with pancreatic lipase activity inhibition function6-ethyl acetate-5' -ethyl acetate-beta-riboadenosine is isolated from culture of Cordyceps cicadae (Cordyceps chanhua) and has the following structural formula:
said N is6The-ethyl acetate-5' -ethyl ester-beta-ribose adenosine is yellow oil, and the molecular formula is C16H21N5O7(ii) a Half maximal Inhibitory Concentration (IC) on pancreatic lipase50) Comprises the following steps: 0.374 mg/mL.
N with pancreatic lipase inhibitory activity6Application of-ethyl acetate-5' -ethyl ester-beta-ribose adenosine in preparing health care products with pancreatic lipase inhibition activity.
N with pancreatic lipase activity inhibition function6The preparation operation steps of the-ethyl acetate-5' -ethyl acetate-beta-ribose adenosine are as follows:
international pool number of cordyceps sobolifera strain used: WDCM 1031;
(1) preparation of solid cultures
(1.1) slant seed culture
Inoculating the cordyceps sobolifera strain water solution stock to a Sasa (SDAY) solid plate culture medium, inoculating 100-300 mu L of the cordyceps sobolifera strain water solution stock to each culture dish, and culturing at a constant temperature of 22-29 ℃ for 8-16 days 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 water is added to the medium to reach the constant volume of 1000 mL;
(1.2) Secondary liquid seed culture
Subjecting first-class bacteriaThe mass of the strain is 0.5-2 cm2100mL of the culture medium is scattered and inoculated into a liquid shake flask culture medium for culture;
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 the triangular flask into the triangular flask with the volume of more than or equal to 100mL, and inoculating 0.5-2 cm into each triangular flask2Performing constant-temperature shaking culture on a 100mL solid flat plate bacterium block at the temperature of 22-29 ℃ and the rotating speed of 180-240 rpm for 3-5 days to obtain a secondary liquid strain;
(1.3) solid culture
Inoculating the secondary liquid seeds to a Sasa (SDAY) solid culture medium, wherein the inoculation amount is that each culture dish is inoculated with 150-300 mu L of secondary liquid seeds, the secondary liquid seeds are uniformly coated, the secondary liquid seeds are placed into a constant-temperature incubator at the temperature of 22-29 ℃ for culture for 4-8 days, and hyphae are collected to obtain a solid culture;
(2) preparation of N6-ethyl acetate-5' -ethyl ester-beta-riboadenosine
(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 of the active ingredient
Primarily purifying the extract by adopting semi-preparative reverse phase high performance liquid chromatography to obtain a yellow-brown solid primary purified substance;
(2.3) purification of the preliminary purified product
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-ethyl acetate-5' -ethyl acetate-beta-riboadenosine, removing solvent under reduced pressure, and drying to obtain yellow oily N6-ethyl acetate-5' -ethyl ester- β -riboadenosine.
The preparation protocol is further defined as follows:
in the step (2.1), the solid culture is dried at the temperature of-50 to 130 ℃, and the weight-volume ratio is 1 g: adding 0.5-5 mL of ethyl acetate extractant into the solid culture, 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 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 (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; collected by peak and divided into 8 fractions, of which the desired compound is present in the 5 th fraction; 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 powdery 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 mobile phase B15% to 30% 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-ethyl acetate-5' -ethyl acetate-beta-riboadenosine, removing solvent under reduced pressure, and drying to obtain yellow oily N6-ethyl acetate-5' -ethyl ester- 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, N6-ethyl acetate-5' -acetyl- β -riboadenosine.
2. The activity test finds that N6Half Inhibitory Concentration (IC) of-acetoxy-5' -acetyl-beta-riboadenosine on pancreatic lipase50) Comprises the following steps: 0.374 mg/ml.
3. Chemical Structure analysis of active Compounds
High resolution LC-MS analysis shows N6-ethyl acetate-5' -acetyl- β -riboadenosine, positive 396.1516(M + H), negative 394.1436(M-H), calculated as C16H21N5O7；
Nmr analysis data are shown in the table below.
The chemical structural formula is obtained by comprehensive liquid chromatography-mass spectrometry analysis and nuclear magnetic resonance analysis, and the chemical structural formula is as follows:
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-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;
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
N6The preparation operation steps of the-ethyl acetate-5' -ethyl acetate-beta-ribose adenosine are as follows:
the international bacteria library number of the used cordyceps sobolifera strain is WDCM 1031.
(1) Preparation of solid cultures
The liquid-solid mixed culture process is adopted, and the specific operation is as follows:
(1.1) slant seed culture
Inoculating Cordyceps cicadae strain water liquid stock seed to Sachs (SDAY) solid plate culture medium, culturing in each culture dish at constant temperature of 29 deg.C for 16d to obtain first-class strain;
a Saxashi (SDAY) solid plate culture medium is prepared from 40g of glucose, 10g of peptone, 10g of yeast extract powder, 20g of agar and water to reach a constant volume of 1000 mL.
(1.2) Secondary liquid seed culture
The first-class strain block is divided into 0.5cm2Breaking 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 50g/L glucose, 12g/L peptone, 12g/L yeast extract powder and distilled water to constant volume;
a500 mL Erlenmeyer flask was charged with 50% Erlenmeyer flask volume of liquid shake flask medium, and each Erlenmeyer flask was inoculated with 1.25cm of solid flat plate mass2Placing the seeds in a constant-temperature oscillation incubator, and culturing for 5d at the temperature of 29 ℃ and the rotating speed of 240rpm 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 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.
(2) Preparation of N6-ethyl acetate-5' -ethyl ester-beta-riboadenosine
(2.1) extraction of effective Components from 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; distilling the filtrate under reduced pressure at 60 deg.C under vacuum degree of-0.1 MP to remove ethyl acetate 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 5-60% 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; collected by peak and divided into 8 fractions, of which the desired compound was present in the 5 th fraction. Distilling off the ethyl acetate extractant under reduced pressure at the vacuum degree of-0.1 MP and the temperature of 60 ℃, and drying at the temperature of 130 ℃ to obtain a yellowish-brown 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 mobile phase B15% to 30% 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-ethyl acetate-5' -ethyl acetate-beta-riboadenosine, removing solvent under reduced pressure, and drying to obtain yellow oily N6-ethyl acetate-5' -ethyl ester- β -riboadenosine.
The purity is over 90 percent by HPLC-MS analysis. The structure is as follows:
from cicada fungus extractA purified nucleoside compound N6Activity experiments show that the compound has obvious inhibition effect on pancreatic lipase activity and half Inhibition Concentration (IC)50) It was 0.374 mg/mL.
Example 2
N6The preparation operation steps of the-ethyl acetate-5' -ethyl acetate-beta-ribose adenosine are as follows:
the international bacteria library number of the used cordyceps sobolifera strain is WDCM 1031.
(1) Preparation of solid cultures
The liquid-solid mixed culture process is adopted, and the specific operation is as follows:
(1.1) slant seed culture
Inoculating Cordyceps cicadae strain water solution stock strain 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;
(1.2) Secondary liquid seed culture
The first-class strain block is divided into 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 shake flask 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;
(1.3) 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.
(2) Preparation of N6-ethyl acetate-5' -ethyl ester-beta-riboadenosine
(2.1) extraction of effective Components from 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; centrifuging at 4000 rpm to obtain a centrifugal supernatant; centrifuging the supernatant, and evaporating 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 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 fraction 5. 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.
(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 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-5' -ethyl acetate-beta-riboadenosine, removing solvent under reduced pressure, and drying to obtain yellow oily N6-ethyl acetate-5' -ethyl ester- 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-ethyl acetate-5' -ethyl ester- β -riboadenosine. The activity experiment shows that the compound has obvious inhibition effect on the activity of pancreatic lipase and half Inhibition Concentration (IC)50) It was 0.374 mg/mL.
Example 3
N6The preparation operation steps of the-ethyl acetate-5' -ethyl acetate-beta-ribose adenosine are as follows:
the international bacteria library number of the used cordyceps sobolifera strain is WDCM 1031.
(1) Preparation of solid cultures
The liquid-solid mixed culture process is adopted, and the specific operation is as follows:
(1.1) slant 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;
(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;
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 fungus2After 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, 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.
(2) Preparation of N6-ethyl acetate-5' -ethyl ester-beta-riboadenosine
(2.1) extraction of effective Components from 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.
(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 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 with the desired compound in fraction 5. 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.
(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 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-5' -ethyl acetate-beta-riboadenosine, removing solvent under reduced pressure, and drying to obtain yellow oily N6-ethyl acetate-5' -ethyl ester- 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 extract6-ethyl acetate-5' -ethyl ester- β -riboadenosine. The activity experiment shows that the compound has obvious inhibition effect on the activity of pancreatic lipase and half Inhibition Concentration (IC)50) It was 0.374 mg/mL.
Example 4
N6The preparation operation steps of the-ethyl acetate-5' -ethyl acetate-beta-ribose adenosine are as follows:
the international bacteria library number of the used cordyceps sobolifera strain is WDCM 1031.
(1) Preparation of solid cultures
The liquid-solid mixed culture process is adopted, and the specific operation is as follows:
(1.1) slant 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;
(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;
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;
a liquid shake flask culture medium of 40% of the volume of a flask was placed in 250mL flasks, and each flask was inoculated with a 1cm solid plate of a fungal mass2Placing the mixture in a constant-temperature oscillation incubator, and culturing for 4d at the temperature of 25 ℃ and the rotating speed of 200rpm to obtain a secondary strain;
(1.3) 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.
(2) Preparation of N6-ethyl acetate-5' -ethyl ester-beta-riboadenosine
(2.1) extraction of effective Components from 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; filtering with 0.2 μm filter membrane to obtain filtrate; evaporating the filtrate under reduced pressure at 40 deg.C under vacuum degree of-0.09 MP to remove the 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 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 with the desired compound in fraction 5. 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.
(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 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-5' -ethyl acetate-beta-riboadenosine, removing solvent under reduced pressure, and drying to obtain yellow oily N6-ethyl acetate-5' -ethyl ester- 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 extract6-ethyl acetate-5' -ethyl ester- β -riboadenosine. The activity experiment shows that the compound has obvious inhibition effect on the activity of pancreatic lipase and half Inhibition Concentration (IC)50) It was 0.374 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 (6)
1. N with pancreatic lipase activity inhibition function6-ethyl acetate-5' -ethyl acetate- β -riboadenosine, characterized by:
is prepared by separating Cordyceps sobolifera (Cordyceps chanhua) culture, and has the following structural formula:
said N is6The-ethyl acetate-5' -ethyl ester-beta-ribose adenosine is yellow oil, and the molecular formula is C16H21N5O7(ii) a Half maximal Inhibitory Concentration (IC) on pancreatic lipase50) Comprises the following steps: 0.374 mg/mL.
2. N having pancrelipase inhibitory activity according to claim 16-use of ethyl acetate-5' -ethyl acetate- β -riboadenosine, characterized in that: said N6Application of-ethyl acetate-5' -ethyl ester-beta-ribose adenosine in preparing health care products for inhibiting pancreatic lipase activity.
3. N having pancrelipase activity inhibiting activity according to claim 16The preparation method of the-ethyl acetate-5' -ethyl acetate-beta-ribose adenosine is characterized by comprising the following operation steps:
international pool number of cordyceps sobolifera strain used: WDCM 1031;
(1) preparation of solid cultures
(1.1) slant seed culture
Inoculating the cordyceps sobolifera strain water solution stock to a Sasa (SDAY) solid plate culture medium, inoculating 100-300 mu L of the cordyceps sobolifera strain water solution stock to each culture dish, and culturing at a constant temperature of 22-29 ℃ for 8-16 days 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 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 cm2100mL of the culture medium is scattered and inoculated into a liquid shake flask culture medium for culture;
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;
at or aboveAdding a liquid culture medium with the volume of 30-50% of that of the triangular flask into a 100mL triangular flask, and inoculating 0.5-2 cm of the liquid culture medium into each triangular flask2Performing constant-temperature shaking culture on a 100mL solid flat plate bacterium block at the temperature of 22-29 ℃ and the rotating speed of 180-240 rpm for 3-5 days to obtain a secondary liquid strain;
(1.3) solid culture
Inoculating the secondary liquid seeds to a Sasa (SDAY) solid culture medium, wherein the inoculation amount is that each culture dish is inoculated with 150-300 mu L of secondary liquid seeds, the secondary liquid seeds are uniformly coated, the secondary liquid seeds are placed into a constant-temperature incubator at the temperature of 22-29 ℃ for culture for 4-8 days, and hyphae are collected to obtain a solid culture;
(2) preparation of N6-ethyl acetate-5' -ethyl ester-beta-riboadenosine
(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 of the active ingredient
Primarily purifying the extract by adopting semi-preparative reverse phase high performance liquid chromatography to obtain a yellow-brown solid primary purified substance;
(2.3) purification of the preliminary purified product
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-ethyl acetate-5' -ethyl acetate-beta-riboadenosine, removing solvent under reduced pressure, and drying to obtain yellow oily N6-ethyl acetate-5' -ethyl ester- β -riboadenosine.
4. The production method according to claim 3, characterized in that: in the step (2.1), the solid culture is dried at the temperature of-50 to 130 ℃, and the weight-volume ratio is 1 g: adding 0.5-5 mL of ethyl acetate extractant into the solid culture, 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 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.
5. The production method according to claim 3, characterized in that: 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; collected by peak and divided into 8 fractions, of which the desired compound is present in the 5 th fraction; 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 powdery primary purified product.
6. The production method according to claim 3, characterized in that: 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 mobile phase B15% to 30% 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-ethyl acetate-5' -ethyl acetate-beta-riboadenosine, removing solvent under reduced pressure, and drying to obtain yellow oily N6-ethyl acetate-5' -ethyl ester- β -riboadenosine.
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| Title |
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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 * |
| 张文轩等: "新型降血脂候选药物WS070117 杂质合成研究", 《中国新药杂志》, vol. 24, no. 10, pages 1166 - 1170 * |
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