Four kinds of Cicadae cicadae nucleosides capable of promoting the proliferation of macrophages and kidney cells and preparation methods thereof

Abstract

The present invention relates to four kinds of cicada flower nucleosides having the activity of promoting the proliferation of macrophages and kidney cells and a preparation method, and belongs to the technical field of extraction and preparation of biological products. The four kinds of cicada flower nucleosides are cicada flower nucleoside A, cicada flower nucleoside B, cicada flower nucleoside C and cicada flower nucleoside D, all of which are yellow oily substances. The structural formulas of the four kinds of cicada flower nucleosides are obtained respectively through one-dimensional and two-dimensional nuclear magnetic resonance and accurate mass spectrometry identification. The present invention obtains compounds of the four kinds of cicada flower nucleosides by culturing cicada flower fungi and then extracting and separating them. The four kinds of cicada flower nucleosides prepared by the present invention can be used to prepare immunostimulants and kidney strengthening preparations, and have potential therapeutic effects on immunosuppression and kidney disease. The preparation method of the present invention comprises preparing solid culture by liquid-solid mixed culture, extracting, preliminarily purifying and refining effective components in the solid culture; the preparation method is environmentally friendly, not affected by the natural environment and resources, easy to realize industrialized, automated and continuous production, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of extraction and preparation of biological products, and specifically relates to four kinds of cicada cordifolia nucleosides capable of promoting the proliferation of macrophages and kidney cells and their extraction, purification, structural identification and activity determination. Background Art

[0002] The four kinds of Cordyceps chanhua nucleosides are Cordyceps chanhua nucleoside A, Cordyceps chanhua nucleoside B, Cordyceps chanhua nucleoside C and Cordyceps chanhua nucleoside D. All of the four kinds of Cordyceps chanhua nucleosides are isolated and prepared from a culture of Cordyceps chanhua fungus and are new biologically active substances that can promote the proliferation of macrophages and kidney cells.

[0003] Cordyceps chanhua is a fungus of the Clavicipitaceae family and the genus Cordyceps. It has many synonyms: Cordyceps cicadae, Cordyceps sinclairii, Isaria sinclairii, Paecilomyces cicadae and Isaria cicadae. It is a Cordyceps complex formed after the Cordyceps chanhua infects insects and is an alternative to Cordyceps sinensis, with important economic value.

[0004] Macrophages are immune cells. Promoting macrophage proliferation can enhance immunity; promoting kidney cell proliferation may play a role in kidney damage repair and kidney disease treatment. Therefore, the four cicadae nucleosides that promote macrophage and kidney cell proliferation have broad application potential in medicine and health products. Summary of the invention

[0005] In order to find new natural and efficient substances with immune-enhancing and kidney-strengthening effects, the inventors conducted activity studies on the metabolites of the cicada fungus RECF5833 strain and discovered that new compounds, cicada fungus nucleoside A, cicada fungus nucleoside B, cicada fungus nucleoside C and cicada fungus nucleoside D, extracted from the cicada fungus culture have a significant effect in promoting the proliferation of macrophages and kidney cells.

[0006] Chanhuauncleosides A, which promotes the proliferation of macrophages and kidney cells, is isolated and prepared from the culture of Cordyceps chanhua, and its structural formula is as follows:

[0007]

[0008] The cicada uncleoside A is a yellow oily substance, and its molecular formula is C 14H 19 N5O6; at a concentration of 50.0 μg / mL, it can accelerate the proliferation of mouse ovarian macrophages by 12.7 times and kidney cells by 5.0 times;

[0009] The Cicadae Cordyceps nucleoside A has application potential in preparing health products that can promote immunity and kidney function.

[0010] Chanhua uncleosides B, which has the function of promoting the proliferation of macrophages and kidney cells, is isolated and prepared from the culture of Cordyceps chanhua, and its structural formula is as follows:

[0011]

[0012] The cicada uncleoside B is a yellow oily substance, and its molecular formula is C 16 H 21 N5O7; at a concentration of 50.0 μg / mL, it can accelerate the proliferation of mouse ovarian macrophages by 13.1 times and kidney cells by 5.5 times;

[0013] The Cicadae riboside B has application potential in preparing health products that can promote immunity and kidney function.

[0014] Chanhuauncleosides C, which promotes the proliferation of macrophages and kidney cells, is isolated and prepared from the culture of Cordyceps chanhua, and its structural formula is as follows:

[0015]

[0016] The cicada uncleoside C is a yellow oily substance, and its molecular formula is C 16 H 21 N5O7; at a concentration of 50.0 μg / mL, it can accelerate the proliferation of mouse ovarian macrophages by 12.5 times and kidney cells by 5.3 times;

[0017] The Cicadae Cordyceps nucleoside C has application potential in preparing health products that can promote immunity and kidney function.

[0018] Chanhuauncleosides D, which promotes the proliferation of macrophages and kidney cells, is isolated and prepared from the culture of Cordyceps chanhua, and its structural formula is as follows:

[0019]

[0020] The cicada uncleoside D is a yellow oily substance, and its molecular formula is C 16 H 21 N5O7; at a concentration of 50.0 μg / mL, it can accelerate the proliferation of mouse ovarian macrophages by 12.2 times and kidney cells by 4.9 times;

[0021] The Cicadae Cordyceps nucleoside D has application potential in preparing health products that can promote immunity and kidney function.

[0022] The preparation steps of four kinds of Cicadae nucleosides that promote the proliferation of macrophages and kidney cells are as follows:

[0023] The international fungus library number of the Cicadae confusa strain used is: WDCM1031;

[0024] (1) Preparation of solid culture

[0025] (1.1) Slant seed culture

[0026] The aqueous stock of the cicada fungus strain was inoculated into the SDAY solid plate culture medium, and each culture dish was inoculated with 100-300 μl of the aqueous stock of the cicada fungus strain, and cultured at a constant temperature of 22-29° C. for 8-16 days to obtain the primary strain;

[0027] The formula of Sabouraud (SDAY) solid plate medium is 40g glucose, 10g peptone, 10g yeast extract powder and 20g agar, add water to make up to 1000mL;

[0028] (1.2) Secondary liquid seed culture

[0029] Press the primary strain into 0.5-2cm thick blocks. 2 / 100mL was broken up and inoculated into liquid shake flask culture medium for cultivation;

[0030] Liquid shake flask culture medium: glucose 30-50 g / L, peptone 8-12 g / L, yeast extract powder 8-12 g / L, distilled water to volume;

[0031] Add 30-50% of the volume of liquid culture medium to a 100 mL flask, and inoculate 0.5-2 cm in each flask. 2 / 100mL solid plate bacteria block, constant temperature shaking culture, temperature 22 ~ 29 ° C, speed 180 ~ 240rpm, culture for 3 ~ 5 days, to obtain the secondary liquid bacteria;

[0032] (1.3) Solid culture

[0033] The secondary liquid seeds are inoculated onto the SDAY solid culture medium, with the inoculation amount being 150 to 300 μL of the secondary liquid seeds per culture dish, spread evenly, and cultured in a constant temperature incubator at 22 to 29° C. for 4 to 8 days, and the mycelium is collected to obtain a solid culture medium;

[0034] (2) Preparation of four kinds of cicada nucleosides

[0035] (2.1) Extraction of active ingredients from solid culture

[0036] The solid culture is subjected to ultrasonic extraction, membrane filtration or centrifugal separation, and vacuum decompression to remove the solvent to obtain an effective component extract using an ethyl acetate extractant;

[0037] (2.2) Preliminary purification of active ingredient extract

[0038] The active ingredient extracts were preliminarily purified using a preparative reverse-phase high performance liquid chromatography device to obtain yellow-brown solid Cordyceps sinensis nucleoside preliminarily purified crude product A, preliminarily purified crude product B, preliminarily purified crude product C and preliminarily purified crude product D, respectively;

[0039] (2.3) Purification of crude product

[0040] The yellow-brown solid cicada nucleoside crude product A, crude product B, crude product C and crude product D were purified respectively by a semi-preparative reverse-phase high performance liquid chromatography apparatus, and under the elution condition of acetonitrile solution, cicada nucleoside A eluate, cicada nucleoside B eluate, cicada nucleoside C eluate and cicada nucleoside D eluate were obtained respectively. The solvent was removed under vacuum decompression and dried to obtain yellow oily cicada nucleoside A, cicada nucleoside B, cicada nucleoside C and cicada nucleoside D respectively.

[0041] The further preparation technology scheme is as follows:

[0042] In step (2.1), the solid culture is dried at a temperature of -50 to 130°C, ethyl acetate extractant is added to the solid culture at a weight volume ratio of 1g:0.5 to 5mL, and ultrasonic extraction is performed at 40KHz for 20 to 200 minutes; filtration is performed through a 0.20 to 2.5μm filter membrane or centrifugation is performed at 4000 to 15000 rpm to obtain a filtrate or a centrifugal supernatant; the filtrate or the centrifugal supernatant is evaporated under reduced pressure at a vacuum degree of -0.1 to -0.09MP and a temperature of 10 to 60°C to obtain an effective component extract.

[0043] In step (2.2), the chromatographic separation preparation conditions are: mobile phase A is ultrapure water, and mobile phase B is acetonitrile; elution conditions: elution with 5% to 60% mobile phase B from 0 to 20min; injection volume is 20-100μL; column temperature is 20-40℃; flow rate is 5-25mL / min; detection is performed with a mass spectrometer in cation mode; the chromatographic column is a preparative reverse phase C-18 column; the eluents corresponding to the mass-to-charge ratio (m / z) of 354 and 396 peaks are collected respectively; under the conditions of vacuum degree -0.1 to -0.08MP and temperature 10 to 60℃, the extractant is removed by reduced pressure evaporation; and drying is performed at a temperature of less than or equal to 130℃ to obtain five yellow-brown components, wherein the second component, the third component, the fourth component and the fifth component are the preliminary purified crude A, preliminary purified crude B, preliminary purified crude C and preliminary purified crude D of cicada flower nucleoside, respectively.

[0044] In step (2.3), the chromatographic separation preparation conditions are: mobile phase A is ultrapure water, and mobile phase B is acetonitrile; elution conditions: elution with 15% to 30% mobile phase B from 0 to 20 min; injection volume 20-100 μL; column temperature 20-40° C.; flow rate 2-5 mL / min; the chromatographic column is a semi-preparative reverse phase C-18 column; mass spectrometry is used for detection in positive ion mode, and the eluents corresponding to the mass-to-charge ratio (m / z) 354 and 396 peaks are collected respectively, and the extractant is removed by reduced pressure evaporation; and drying is carried out at a temperature of less than or equal to 130° C. to obtain yellow oily Cordyceps nucleoside A, Cordyceps nucleoside B, Cordyceps nucleoside C and Cordyceps nucleoside D respectively.

[0045] The analytical research of the present invention is described as follows:

[0046] 1. The ethyl acetate extract of Cicadae Mycelia was separated and purified by high performance liquid chromatography to obtain four new biologically active compounds, namely Cicadae Nucleoside A, Cicadae Nucleoside B, Cicadae Nucleoside C and Cicadae Nucleoside D.

[0047] 2. Activity tests found that Cicada nucleoside A at a concentration of 50.0 μg / mL can accelerate the proliferation of mouse ovarian macrophages by 12.7 times and kidney cells by 5.0 times; Cicada nucleoside B at a concentration of 50.0 μg / mL can accelerate the proliferation of mouse ovarian macrophages by 13.1 times and kidney cells by 5.5 times; Cicada nucleoside C at a concentration of 50.0 μg / mL can accelerate the proliferation of mouse ovarian macrophages by 12.5 times and kidney cells by 5.3 times. Cicada nucleoside D at a concentration of 50.0 μg / mL can accelerate the proliferation of mouse ovarian macrophages by 12.2 times and kidney cells by 4.9 times.

[0048] 3. Chemical structure analysis of active compounds

[0049] (1) High-resolution LC-MS analysis showed that the positive ion of Cicadae nucleoside A was 354.1472 (M+H) and the negative ion was 352.1396 (MH). The calculated molecular formula was C 14 H 19 N5O6;

[0050] The NMR analysis data are shown in Table 1 below.

[0051]

[0052] (2) High-resolution LC-MS analysis showed that the positive ion of Cicadae denucleoside B was 396.1516 (M+H) and the negative ion was 394.1436 (MH). The calculated molecular formula was C 16 H 21 N5O7;

[0053] The NMR analysis data are shown in Table 2 below.

[0054]

[0055]

[0056] (3) High-resolution LC-MS analysis showed that the positive ion of Cicadae nucleoside C was 396.1517 (M+H) and the negative ion was 394.1438 (MH). The calculated molecular formula was C 16 H 21 N5O7;

[0057] The NMR analysis data are shown in Table 3 below.

[0058]

[0059] (4) High-resolution LC-MS analysis showed that the positive ion of Cicadae denucleoside D was 396.1517 (M+H) and the negative ion was 394.1438 (MH). The calculated molecular formula was C 16 H 21 N5O7;

[0060] The NMR analysis data are shown in Table 4 below.

[0061]

[0062]

[0063] The chemical structure obtained by comprehensive LC-MS analysis and NMR analysis is as follows:

[0064]

[0065] The beneficial technical effects of the present invention are embodied in the following aspects:

[0066] 1. In order to find new substances with the activity of promoting the proliferation of macrophages and kidney cells, the inventors first cultured a large number of Cicadae confusa strains, then extracted and tested the active ingredients of the culture, and found that an extract of Cicadae confusa culture had the activity of promoting the proliferation of macrophages and kidney cells. On the basis of large-scale culture and extraction, four new active compounds, Cicadae confusa nucleoside A, Cicadae confusa nucleoside B, Cicadae confusa nucleoside C and Cicadae confusa nucleoside D, were separated and purified by semi-preparative chromatography. These compounds have strong activity in promoting the proliferation of macrophages and kidney cells, and can play a role in enhancing immunity, repairing kidney damage and treating kidney disease, opening up a new application field for Cicadae confusa.

[0067] 2. The macrophage and kidney cell proliferation promoter prepared by the present invention has a wide range of uses and can be used as a medical drug to reduce lipids, enhance immunity, and treat kidney disease.

[0068] 3. The present invention adopts microbial fermentation production, which is not affected by the environment and resources, and is easy to realize industrialization and automation, and is not affected by the environment and natural resources.

[0069] 4. The products produced by the process method of the present invention have low cost, simple process, stable process, easy regulation and control, and high success rate; the investment in production equipment can be large or small, the production is flexible, and it is suitable for investment by enterprises of various sizes. DETAILED DESCRIPTION

[0070] The present invention will be further described below in conjunction with embodiments.

[0071] Example 1

[0072] The preparation steps of four kinds of cicada nucleosides are as follows:

[0073] The strain number of the Cordyceps chanhua used is RCEF5833, which is preserved in the Research Center on Entomogenous Fungi (RCEF) of Anhui Agricultural University, and its international fungus library number is WDCM1031.

[0074] (1) Preparation of solid culture

[0075] The liquid-solid mixed culture process is adopted, and the specific operations are as follows:

[0076] (1.1) Slant seed culture

[0077] The original water stock of Cicadae confusa strain was inoculated into SDAY solid plate medium, 300 μL per culture dish, and cultured at 29°C for 16 days to obtain the primary strain.

[0078] Sabouraud (SDAY) solid plate medium consists of 40 g of glucose, 10 g of peptone, 10 g of yeast extract powder, 20 g of agar, and water is added to make up to 1000 mL.

[0079] (1.2) Secondary liquid seed culture

[0080] Press the first-level bacterial strain block into 0.5cm 2 / 100mL was broken up and inoculated into liquid shake flask culture medium for cultivation;

[0081] Liquid shake flask medium consists of 50 g / L glucose, 12 g / L peptone, 12 g / L yeast extract powder, and distilled water to volume;

[0082] Add 50% of the volume of the liquid shake flask medium to a 500 mL flask and inoculate each flask with a 1.25 cm solid plate bacterial block. 2 , placed in a constant temperature shaking incubator at 29°C and 240rpm, cultured for 5 days to obtain secondary liquid seeds.

[0083] (1.3) Solid culture

[0084] The secondary liquid seeds were inoculated onto the Sabouraud (SDAY) solid culture medium with an inoculation volume of 300 μL per culture dish, spread evenly, and cultured in a 29° C. constant temperature incubator for 8 days. Mycelium was collected to obtain a solid culture.

[0085] (2) Preparation of four kinds of cicada nucleosides

[0086] (2.1) Extraction of active ingredients from solid culture

[0087] The solid culture was dried at a temperature of 130°C, and ethyl acetate extractant was added to the solid culture at a weight volume ratio of 1g:5mL, and ultrasonic extraction was performed at 40KHz for 200 minutes; the filtrate was filtered through a 2.5μm filter membrane to obtain a filtrate; the filtrate was evaporated under reduced pressure to remove the ethyl acetate extractant at a vacuum degree of -0.1MP and a temperature of 60°C to obtain an effective component extract.

[0088] (2.2) Preliminary purification of active ingredient extracts

[0089] The effective component extract was preliminarily purified using a preparative reversed-phase high performance liquid chromatography device. The chromatographic separation preparation conditions were as follows: mobile phase A was ultrapure water, mobile phase B was acetonitrile; elution conditions: elution with 5-60% mobile phase B from 0 to 20 min; injection volume was 100 μL; column temperature was 40°C; flow rate was 25 mL / min; detection was performed using a mass spectrometer in the cation mode; the chromatographic column was a preparative reversed-phase C-18 column; the eluents corresponding to the peaks of mass-to-charge ratio (m / z) of 354 and 396 were collected respectively; the ethyl acetate extractant was evaporated under reduced pressure at a vacuum degree of -0.1 MP and a temperature of 60°C, and dried at 130°C to obtain five yellow-brown components, of which the second component, the third component, the fourth component, and the fifth component were the preliminary purified crude A, preliminary purified crude B, preliminary purified crude C, and preliminary purified crude D of Cordyceps sinensis nucleoside, respectively.

[0090] (2.3) Purification of crude product

[0091] The semi-preparative reversed-phase high performance liquid chromatography device was used to purify the preliminary purified crude A, preliminary purified crude B, preliminary purified crude C and preliminary purified crude D of Cicadella nucleoside. The chromatographic separation preparation conditions were as follows: mobile phase A was ultrapure water, mobile phase B was acetonitrile; elution conditions: elution with 15% to 30% mobile phase B from 0 to 20min; injection volume was 100μL; column temperature was 40℃; flow rate was 5mL / min; mass spectrometry detection, the chromatographic column was a reversed-phase C-18 column; the eluents corresponding to the mass-to-charge ratio (m / z) 354 and 396 peaks were collected respectively, and the extractant was removed by evaporation under reduced pressure; and dried at 130℃ to obtain yellow oily Cicadella nucleoside A, Cicadella nucleoside B, Cicadella nucleoside C and Cicadella nucleoside D respectively.

[0092] The purity of the product is above 95% as determined by HPLC-MS. Its structure is as follows according to NMR analysis:

[0093]

[0094] The biological activity analysis found that Cicadae riboside A at a concentration of 50.0 μg / mL could accelerate the proliferation of mouse ovarian macrophages by 12.7 times and kidney cells by 5.0 times;

[0095] At a concentration of 50.0 μg / mL, Cicadae riboside B can accelerate the proliferation of mouse ovarian macrophages by 13.1 times and kidney cells by 5.5 times;

[0096] At a concentration of 50.0 μg / mL, Cicadae riboside C can accelerate the proliferation of mouse ovarian macrophages by 12.5 times and kidney cells by 5.3 times;

[0097] At a concentration of 50.0 μg / mL, cicadacarposide D can accelerate the proliferation of mouse ovarian macrophages by 12.2 times and the proliferation of renal cells by 4.9 times.

[0098] Example 2

[0099] The preparation steps of four kinds of cicada nucleosides are as follows:

[0100] The international fungus library number of the Cicadae confusa strain used is WDCM1031, the same as in Example 1.

[0101] (1) Preparation of solid culture

[0102] The liquid-solid mixed culture process is adopted, and the specific operations are as follows:

[0103] (1.1) Slant seed culture

[0104] The aqueous stock of the Cordyceps sinensis strain was inoculated on the SDAY solid plate medium, with 100 μL inoculated on each plate, and cultured at 22°C for 8 days to obtain the primary strain.

[0105] (1.2) Secondary liquid seed culture

[0106] Press the first-level bacterial strain block into 2cm 2 / 100mL was broken up and inoculated into liquid shake flask culture medium for cultivation;

[0107] Liquid shake flask medium consists of 30 g / L glucose, 8 g / L peptone, 8 g / L yeast extract powder, and distilled water to volume;

[0108] Add 30% of the volume of the liquid shake flask medium to a 100 mL flask and inoculate each flask with a solid plate 0.6 cm 2 The bacterial block was placed in a constant temperature shaking incubator at 22°C and 180 rpm for 3 days to obtain the secondary liquid seed;

[0109] (1.3) Solid culture

[0110] The secondary liquid seeds were inoculated onto the Sabouraud (SDAY) solid culture medium with an inoculation volume of 150 μL per culture dish, spread evenly, and cultured in a 22° C. constant temperature incubator for 4 days. Mycelium was collected to obtain a solid culture.

[0111] (2) Preparation of Cicadae cicadae nucleosides A, B, C and D

[0112] (2.1) Extraction of active ingredients from solid culture

[0113] The solid culture was dried at a temperature of -50°C, and an ethyl acetate extractant was added to the solid culture at a weight volume ratio of 1g:0.5mL, and ultrasonic extraction was performed at 40KHz for 20 minutes; the solid culture was centrifuged at 4000 rpm to obtain a centrifugal supernatant; the centrifugal supernatant was evaporated under reduced pressure at a vacuum degree of -0.08MP and a temperature of 10°C to obtain an effective component extract.

[0114] (2.2) Preliminary purification of active ingredient extracts

[0115] The effective component extract was preliminarily purified using a preparative reversed-phase high performance liquid chromatography device. The chromatographic separation preparation conditions were: semi-preparative liquid chromatography, mobile phase A was ultrapure water, and mobile phase B was acetonitrile; elution conditions: elution with 5% to 60% mobile phase B from 0 to 20 min; injection volume 20 μL, column temperature 20°C, flow rate 5 mL / min; detection with a mass spectrometer in cation mode; the chromatographic column was a preparative reversed-phase C-18 column; the eluents corresponding to the peaks of mass-to-charge ratio (m / z) 354 and 396 were collected respectively; the ethyl acetate extractant was evaporated under reduced pressure at a vacuum degree of -0.08 MP and a temperature of 10°C, and dried at -50°C to obtain five yellow-brown components, of which the second component, the third component, the fourth component, and the fifth component were the preliminary purified crude A, preliminary purified crude B, preliminary purified crude C, and preliminary purified crude D of Cordyceps sinensis nucleoside, respectively.

[0116] (2.3) Purification of crude product

[0117] The semi-preparative reversed-phase high performance liquid chromatography device is used to purify the preliminary purified crude A, preliminary purified crude B, preliminary purified crude C and preliminary purified crude D of Cicadella nucleoside. The chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, mobile phase B is acetonitrile; elution conditions: elution with 15% to 30% mobile phase B from 0 to 20min; injection volume is 20μL; column temperature is 20℃; flow rate is 2mL / min; the chromatographic column is a semi-preparative reversed-phase C-18 column; mass spectrometry is used for detection in the positive ion mode, and the eluents corresponding to the mass-to-charge ratio (m / z) 354 and 396 peaks are collected respectively, and the extractant is removed by evaporation under reduced pressure; and dried at -40℃ to obtain yellow oily Cicadella nucleoside A, Cicadella nucleoside B, Cicadella nucleoside C and Cicadella nucleoside D.

[0118] The purity of the product is above 95% as determined by HPLC-MS. Its structure is as follows according to NMR analysis:

[0119]

[0120] The biological activity analysis found that Cicadae riboside A at a concentration of 50.0 μg / mL could accelerate the proliferation of mouse ovarian macrophages by 12.7 times and kidney cells by 5.0 times;

[0121] At a concentration of 50.0 μg / mL, Cicadae riboside B can accelerate the proliferation of mouse ovarian macrophages by 13.1 times and kidney cells by 5.5 times;

[0122] At a concentration of 50.0 μg / mL, Cicadae riboside C can accelerate the proliferation of mouse ovarian macrophages by 12.5 times and kidney cells by 5.3 times;

[0123] At a concentration of 50.0 μg / mL, cicadacarposide D can accelerate the proliferation of mouse ovarian macrophages by 12.2 times and the proliferation of renal cells by 4.9 times.

[0124] Example 3

[0125] The preparation steps of Cicadae nucleosides A, B, C and D are as follows:

[0126] The international fungus library number of the Cicadae confusa strain used is WDCM1031, the same as in Example 1.

[0127] (1) Preparation of solid culture

[0128] The liquid-solid mixed culture process is adopted, and the specific operations are as follows:

[0129] (1.1) Slant seed culture

[0130] The aqueous stock of the Cordyceps sinensis strain was inoculated on a Sabouraud (SDAY) solid plate medium, with 200 μL inoculated on each plate, and cultured at 25°C for 12 days to obtain the primary strain.

[0131] (1.2) Secondary liquid seed culture

[0132] Press the first-level bacterial strain into 1cm 2 / 100mL was broken up and inoculated into liquid shake flask culture medium for cultivation;

[0133] Liquid shake flask medium consists of 40 g / L glucose, 10 g / L peptone, 10 g / L yeast extract powder, and distilled water to volume;

[0134] Add 40% of the volume of liquid culture medium to a 250 mL flask and inoculate each flask with a 1 cm thick solid plate bacterial block. 2 After inoculation, the seeds were placed in a constant temperature shaking incubator at 25°C and 200 rpm for 4 days to obtain secondary liquid seeds.

[0135] (1.3) Solid culture

[0136] The secondary liquid seeds were inoculated onto SDAY solid culture medium with an inoculation volume of 225 μL per culture dish, spread evenly, and cultured in a 25° C. constant temperature incubator for 6 days. Mycelium was collected to obtain solid culture.

[0137] (2) Preparation of Cicadae cicadae nucleosides A, B, C and D

[0138] (2.1) Extraction of active ingredients from solid culture

[0139] The solid culture was dried at 60°C, ethyl acetate extractant was added to the solid culture at a weight volume ratio of 1g:3mL, and ultrasonic extraction was performed at 40KHz for 120 minutes; centrifuged at 15000 rpm to obtain a centrifugal supernatant; the extractant was evaporated under reduced pressure at a vacuum degree of -0.09MP and a temperature of 40°C to obtain an effective component extract.

[0140] (2.2) Preliminary purification of active ingredient extracts

[0141] The effective component extract was preliminarily purified using a preparative reversed-phase high performance liquid chromatography device. The chromatographic separation preparation conditions were as follows: mobile phase A was ultrapure water, and mobile phase B was acetonitrile; elution conditions: elution with 5% to 60% mobile phase B from 0 to 20 min; injection volume was 60 μL, column temperature was 30°C, and flow rate was 15 mL / min; detection was performed using a mass spectrometer in the positive ion mode; the chromatographic column was a preparative reversed-phase C-18 column; the eluents corresponding to the peaks of mass-to-charge ratio (m / z) of 354 and 396 were collected respectively; the ethyl acetate extractant was evaporated under reduced pressure at a vacuum degree of -0.09 MP and a temperature of 40°C, and dried at 100°C to obtain five yellow-brown components, of which the second component, the third component, the fourth component, and the fifth component were the preliminary purified crude A, preliminary purified crude B, preliminary purified crude C, and preliminary purified crude D of the cicada flower nucleoside, respectively.

[0142] (2.3) Purification of crude product

[0143] The semi-preparative reversed-phase high performance liquid chromatography device was used to purify the preliminary purified crude A, preliminary purified crude B, preliminary purified crude C and preliminary purified crude D of Cicadella nucleoside. The chromatographic separation preparation conditions were as follows: mobile phase A was ultrapure water, mobile phase B was acetonitrile; elution conditions: elution with 15% to 30% mobile phase B from 0 to 20 minutes; injection volume was 20 μL; column temperature was 30°C; flow rate was 3.5 mL / min; mass spectrometry was used for detection in the positive ion mode, and the chromatographic column was a reversed-phase C-18 column; the eluents corresponding to the peaks of mass-to-charge ratio (m / z) 354 and 396 were collected respectively, and the extractant was removed by evaporation under reduced pressure; and dried at -40°C to obtain yellow oily Cicadella nucleoside A, Cicadella nucleoside B, Cicadella nucleoside C and Cicadella nucleoside D respectively.

[0144] The purity of the product is above 95% as determined by HPLC-MS. Its structure is as follows according to NMR analysis:

[0145]

[0146] The biological activity analysis found that Cicadae riboside A at a concentration of 50.0 μg / mL could accelerate the proliferation of mouse ovarian macrophages by 12.7 times and kidney cells by 5.0 times;

[0147] At a concentration of 50.0 μg / mL, Cicadae riboside B can accelerate the proliferation of mouse ovarian macrophages by 13.1 times and kidney cells by 5.5 times;

[0148] At a concentration of 50.0 μg / mL, Cicadae riboside C can accelerate the proliferation of mouse ovarian macrophages by 12.5 times and kidney cells by 5.3 times;

[0149] At a concentration of 50.0 μg / mL, cicadacarposide D can accelerate the proliferation of mouse ovarian macrophages by 12.2 times and the proliferation of renal cells by 4.9 times.

[0150] Example 4

[0151] The preparation steps of Cicadae nucleosides A, B, C and D are as follows:

[0152] The international fungus library number of the Cicadae confusa strain used is WDCM1031, the same as in Example 1.

[0153] (1) Preparation of solid culture

[0154] The liquid-solid mixed culture process is adopted, and the specific operations are as follows:

[0155] (1.1) Slant seed culture

[0156] The aqueous stock of the Cordyceps sinensis strain was inoculated on a Sabouraud (SDAY) solid plate medium, with 200 μL inoculated on each plate, and cultured at 25°C for 12 days to obtain the primary strain.

[0157] (1.2) Secondary liquid seed culture

[0158] Press the first-level bacterial strain into 1cm 2 / 100mL was broken up and inoculated into liquid shake flask culture medium for cultivation;

[0159] Liquid shake flask medium consists of 40 g / L glucose, 10 g / L peptone, 10 g / L yeast extract powder, and distilled water to volume;

[0160] Add 40% of the volume of the flask into the liquid shake flask medium in a 250 mL flask, and inoculate each flask with a 1 cm solid plate bacterial block. 2 , placed in a constant temperature shaking incubator at 25°C and 200 rpm, cultured for 4 days to obtain secondary strains;

[0161] (1.3) Solid culture

[0162] The secondary liquid seeds were inoculated onto SDAY solid culture medium with an inoculation volume of 225 μL per culture dish, spread evenly, and cultured in a 25° C. constant temperature incubator for 6 days. Mycelium was collected to obtain solid culture.

[0163] (2) Preparation of four kinds of cicada nucleosides

[0164] (2.1) Extraction of active ingredients from solid culture

[0165] The solid culture was dried at 60°C, ethyl acetate extractant was added to the solid culture at a weight volume ratio of 1g:3mL, and ultrasonic extraction was performed at 40KHz for 120 minutes; the filtrate was filtered through a 0.2μm filter membrane to obtain a filtrate; the filtrate was evaporated under reduced pressure at a vacuum degree of -0.09MP and a temperature of 40°C to remove the extractant to obtain an effective component extract.

[0166] (2.2) Preliminary purification of active ingredient extracts

[0167] The effective component extract was preliminarily purified using a preparative reversed-phase high performance liquid chromatography device. The chromatographic separation preparation conditions were as follows: mobile phase A was ultrapure water, and mobile phase B was acetonitrile; elution conditions: elution with 5% to 60% mobile phase B from 0 to 20 min; injection volume was 60 μL, column temperature was 30°C, and flow rate was 10 mL / min; detection was performed using a mass spectrometer in the positive ion mode; the chromatographic column was a preparative reversed-phase C-18 column; the eluents corresponding to the peaks of mass-to-charge ratio (m / z) of 354 and 396 were collected respectively; under the conditions of vacuum degree -0.09 MP and temperature of 40°C, the ethyl acetate extractant was evaporated under reduced pressure, and dried at 60°C to obtain five yellow-brown components, of which the second component, the third component, the fourth component, and the fifth component were the preliminary purified crude A, preliminary purified crude B, preliminary purified crude C, and preliminary purified crude D of Cordyceps sinensis nucleoside, respectively.

[0168] (2.3) Purification of crude product

[0169] The semi-preparative reversed-phase high performance liquid chromatography device was used to purify the preliminary purified crude A, preliminary purified crude B, preliminary purified crude C and preliminary purified crude D of Cicadella nucleoside. The chromatographic separation preparation conditions were as follows: mobile phase A was ultrapure water, mobile phase B was acetonitrile; elution conditions: elution with 15% to 30% mobile phase B from 0 to 20 minutes; injection volume was 20 μL; column temperature was 20°C; flow rate was 3 mL / min; mass spectrometry detection, the chromatographic column was a semi-preparative reversed-phase C-18 column; the eluents corresponding to the mass-to-charge ratio (m / z) 354 and 396 peaks were collected respectively, and the extractant was removed by evaporation under reduced pressure; and dried at 60°C to obtain yellow oily Cicadella nucleoside A, Cicadella nucleoside B, Cicadella nucleoside C and Cicadella nucleoside D respectively.

[0170] The purity of the product is above 95% as determined by HPLC-MS. Its structure is as follows according to NMR analysis:

[0171]

[0172] The biological activity analysis found that Cicadae riboside A at a concentration of 50.0 μg / mL could accelerate the proliferation of mouse ovarian macrophages by 12.7 times and kidney cells by 5.0 times;

[0173] At a concentration of 50.0 μg / mL, Cicadae riboside B can accelerate the proliferation of mouse ovarian macrophages by 13.1 times and kidney cells by 5.5 times;

[0174] At a concentration of 50.0 μg / mL, Cicadae riboside C can accelerate the proliferation of mouse ovarian macrophages by 12.5 times and kidney cells by 5.3 times;

[0175] At a concentration of 50.0 μg / mL, cicadacarposide D can accelerate the proliferation of mouse ovarian macrophages by 12.2 times and the proliferation of renal cells by 4.9 times.

[0176] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Chanhuauncleosides D, which promotes the proliferation of macrophages and kidney cells, is characterized by: Is from cicada fungus ( Cordyceps chanhua ) culture, and its structural formula is as follows: ； Chanhuauncleoside D R1=acetyl R2=H R3=H R4=acetyl; The cicada uncleoside D is a yellow oily substance, and its molecular formula is C 16 H 21 N5O7; at a concentration of 50.0 μg / mL, it can accelerate the proliferation of mouse ovarian macrophages by 12.2 times and kidney cells by 4.9 times; The Cicadae Cordyceps nucleoside D has application potential in preparing health products that can promote immunity and kidney function.

2. The method for preparing Chanhuauncleosides D that promotes the proliferation of macrophages and kidney cells according to claim 1, characterized in that The steps are as follows: The international fungus library number of the Cicadae confusa strain used is: WDCM1031; (1) Preparation of solid culture (1.1) Slant seed culture The original water stock of the cicada fungus strain was inoculated into the SDAY solid plate culture medium, and each culture dish was inoculated with 100-300 μl of the original water stock of the cicada fungus strain, and cultured at a constant temperature of 22-29°C for 8-16 days to obtain the primary strain; The formula of Sabouraud (SDAY) solid plate medium is 40 g glucose, 10 g peptone, 10 g yeast extract powder and 20 g agar, add water to make up to 1000 mL; (1.2) Secondary liquid seed culture Press the primary strain into 0.5-2cm thick blocks. 2 / 100mL was broken up and inoculated into liquid shake flask culture medium for cultivation; Liquid shake flask culture medium: glucose 30-50 g / L, peptone 8-12 g / L, yeast extract powder 8-12 g / L, distilled water to volume; Add 30-50% of the volume of liquid culture medium to a 100 mL flask, and inoculate 0.5-2 cm 2 / 100mL solid plate bacteria block, constant temperature shaking culture, temperature 22 ~ 29 ° C, speed 180 ~ 240 rpm, culture for 3 ~ 5 days, to obtain the secondary liquid bacteria; (1.3) Solid culture The secondary liquid seeds are inoculated onto the SDAY solid culture medium, and the inoculation amount is 150-300 μL of the secondary liquid seeds per culture dish, and the culture is spread evenly, and the culture is placed in a constant temperature incubator at 22-29° C. for 4-8 days, and the mycelium is collected to obtain a solid culture medium; (2) Preparation of Cicadae Nucleosides (2.1) Extraction of active ingredients from solid culture The solid culture is subjected to ultrasonic extraction, membrane filtration or centrifugal separation, and vacuum decompression to remove the solvent to obtain an effective component extract using an ethyl acetate extractant; (2.2) Preliminary purification of active ingredient extract The active ingredient extract was preliminarily purified using a preparative reverse-phase high performance liquid chromatography device to obtain a yellow-brown solid crude product D of Cicadae denudata nucleoside; (2.3) Refining of crude product The yellow-brown solid Cordyceps militaris nucleoside crude product D was purified by a semi-preparative reverse-phase high performance liquid chromatography device, and Cordyceps militaris nucleoside D eluate was obtained under acetonitrile solution elution conditions. The solvent was removed under vacuum decompression and dried to obtain yellow oily Cordyceps militaris nucleoside D.

3. The preparation method according to claim 2, characterized in that: In step (2.1), the solid culture is dried at a temperature of -50 to 130°C, ethyl acetate extractant is added to the solid culture at a weight volume ratio of 1 g: 0.5 to 5 mL, and ultrasonic extraction is performed at 40 kHz for 20 to 200 minutes; the solid culture is filtered through a 0.20 to 2.5 μm filter membrane or centrifuged at 4000 to 15000 rpm to obtain a filtrate or a centrifugal supernatant; the filtrate or the centrifugal supernatant is evaporated under reduced pressure at a vacuum degree of -0.1 to -0.09 MP and a temperature of 10 to 60°C to remove the extractant to obtain an effective component extract.

4. The preparation method according to claim 2, characterized in that: In step (2.2), the chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, and mobile phase B is acetonitrile; elution conditions: elution with 5% to 60% mobile phase B from 0 to 20 min; injection volume is 20 to 100 μL; column temperature is 20 to 40°C; flow rate is 5 to 25 mL / min; detection is performed using a mass spectrometer in the positive ion mode; the chromatographic column is a preparative reverse phase C-18 column; the eluents corresponding to the peaks with mass-to-charge ratios (m / z) of 354 and 396 are collected respectively; under vacuum conditions of -0.1 to -0.08 MP and temperature of 10 to 60°C, the extractant is removed by reduced pressure evaporation; and drying is performed at a temperature of less than or equal to 130°C to obtain a yellow-brown solid crude product D of preliminary purified cicada cordifolia nucleoside.

5. The preparation method according to claim 2, characterized in that: In step (2.3), the chromatographic separation preparation conditions are as follows: mobile phase A is ultrapure water, and mobile phase B is acetonitrile; elution conditions: elution with 15% to 30% mobile phase B from 0 to 20 min; injection volume is 20-100 μL; column temperature is 20-40°C; flow rate is 2-5 mL / min; the chromatographic column is a semi-preparative reverse phase C-18 column; mass spectrometry is used for detection in positive ion mode, and the eluents corresponding to the mass-to-charge ratio (m / z) 354 and 396 peaks are collected respectively, and the extractant is removed by evaporation under reduced pressure; and drying is performed at a temperature of less than or equal to 130°C to obtain a yellow oily cicada nucleoside D.

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