CN104694616A - Method for detecting content of adenosine and cordycepin in cordyceps militaris mycelium - Google Patents

Abstract

The invention discloses a method for detecting the contents of adenosine and cordycepin in the mycelium of Cordyceps militaris. The method specifically includes the following steps: 1) The mycelium of Cordyceps militaris is repeatedly frozen and thawed and subjected to ultrasonication in a water bath, and the filtrate after ultrasonication is used as the sample to be tested; 2) The sample to be tested obtained in step 1) and adenosine and/or The solution of the cordycepin standard substance is detected by high performance liquid chromatography, and the content of adenosine and/or cordycepin in the sample to be tested is calculated according to the detection results. Compared with the existing method, this method does not need to dry the wet mycelium of liquid fermentation culture, and can simultaneously measure the content of adenosine and cordycepin in the mycelia, and the linear range of the measured adenosine is 2-50 μg/mL , the linear range of cordycepin is 3-50 μg/mL, the detection limits of adenosine and cordycepin are 1.1 μg/mL and 2.4 μg/mL respectively, and the recoveries of the two components are 94%-100% and 99% respectively -103%. This method has the advantages of saving time, energy, and fast analysis.

Description

A method for detecting the content of adenosine and cordycepin in Cordyceps militaris mycelium

technical field

The present invention relates to a method for detecting the content of adenosine and cordycepin in Cordyceps militaris mycelium, in particular to a method for simultaneously determining adenosine and cordycepin in wet mycelium of Cordyceps militaris after ultrasonic treatment by using high performance liquid chromatography content method. the

Background technique

Cordyceps militaris, also known as Cordyceps militaris, is widely distributed in my country. As a medicinal and edible fungus, Cordyceps militaris has been selected into the catalog of new food resources. In recent years, people have isolated a variety of biologically active substances from the fruiting body and mycelia of Cordyceps militaris, mainly including adenosine, cordycepin, cordyceps polysaccharide, D-mannitol, SOD enzyme, sterols and other components, which have anti-inflammatory, Anti-oxidation, anti-tumor, anti-cancer, improving immunity and other physiological functions. Studies have shown that the content of active ingredients such as adenosine and cordycepin in Cordyceps militaris is equivalent to that of wild Cordyceps sinensis, and has broad application prospects. the

At present, the artificial cultivation technology of Cordyceps militaris is relatively mature. According to different cultivation methods, it can be divided into solid cultivation and liquid fermentation. Compared with solid cultivation, liquid fermentation has the advantages of short cycle, simple process, less pollution, and large mycelium output. Etc. As important indicators for the quality evaluation of Cordyceps militaris, adenosine and cordycepin have become important indicator substances for the fermentation and cultivation of Cordyceps militaris mycelia. Therefore, a rapid and effective detection method is of great significance for guiding the research on the liquid fermentation process of Cordyceps militaris. the

At present, the content of adenosine and cordycepin is detected by high performance liquid chromatography, capillary electrophoresis and other methods, while the determination of the content of adenosine and cordycepin in the wet mycelia of Cordyceps militaris is generally carried out by detecting the content of the two components in the dry mycelium However, in the drying process of wet mycelium, there are disadvantages such as long time consumption, high energy consumption, and easy change of ingredients. For example, Niu Shuang et al. determined adenosine in Cordyceps militaris mycelia (NIU Shuang, GAO Xiang, AN Jin-shuang, GUO Jing-jing, TENG Li-rong, LU Jia-hui. Optimization of the Water Extraction Technique of Cordyceps militaris Mycelium Adenosine via Response Surface Methodology[J].Li Shizhen Medicine and Medical Research.2009,20(2):323-324.) Vacuum freeze-drying was used as sample pretreatment, and only one component of adenosine was determined. Adenosine and cordycepin cannot be detected at the same time, and this method is time-consuming and energy-consuming. the

Contents of the invention

The purpose of the present invention is to provide a method for detecting the content of adenosine and cordycepin in the mycelium of Cordyceps militaris. the

The method for detecting the content of adenosine and/or cordycepin in the mycelia of Cordyceps militaris provided by the present invention may specifically include the following steps:

(1) Repeated freezing and thawing of the mycelium of Cordyceps militaris was performed in a water bath for ultrasonication, and the filtrate after ultrasonication was used as the sample to be tested;

(2) The sample to be tested obtained in step (1) and the solution of adenosine and/or cordycepin standard substance were subjected to high-performance liquid chromatography detection under the same conditions, and calculated according to the test results to obtain the The content of adenosine and/or cordycepin. the

In the step (1) of the method, the number of times of repeated freezing and thawing can be 1-3 times (such as 2-3 times); the freezing and thawing time of one time is: freezing in liquid nitrogen for 20-30min, and transferring to Thaw at 50-60°C for 10-15 minutes. Further, for cordycepin, the number of repeated freezing and thawing is preferably 2-3 times. the

In the step (1) of the method, the condition of the water bath ultrasonic is 35-45KHz (eg 40KHz) continuous ultrasonic, the temperature may be 30-50°C (eg 40-50°C), and the time may be 2-4h. Further, for adenosine, the temperature is more preferably 40-50°C. the

In the step (2) of the method, the conditions of the high performance liquid chromatography are as follows: the chromatographic column can be a C18 chromatographic column; the mobile phase is methanol and water according to the volume ratio of (10-20):(80-90) (such as 15:85) mixed solution; the elution method is single mobile phase elution, and the elution time is 15min. the

In the present invention, the chromatographic column is a Klomasail C18 (150mm×4.6mm5μm particle size) chromatographic column. More specifically, it is a product of Sweden AKZO NOBEL company, and its product catalog number is EH04713. the

Further, in step (2), the flow rate of the high performance liquid chromatography may be 1 mL/min, the injection volume may be 20 μL, and the column temperature may be 25° C. the

In the step (1) of the method, the Cordyceps militaris mycelium is specifically obtained according to the following method: Centrifuge 1600-1800g (such as 1700g) of the Cordyceps militaris fermentation liquid for 15-20min, discard the supernatant, and obtain the pupae Cordyceps mycelium. the

The Cordyceps militaris fermentation liquid is obtained by inoculating and culturing the seed liquid of the Cordyceps militaris mycelium (cell concentration: 1 g/100 mL, mycelium mass/bacterial liquid volume) in a fermentation medium. The solvent of the fermentation medium is water, and the solute and its concentration are as follows: 70g/L sucrose, 30g/L soybean peptone, 30g/L yeast extract, 15g/L bean cake powder, K ₂ HPO ₄ ·3H ₂ O2g/ L, MgSO ₄ ·7H ₂ O 1g/L; pH is 7. The culture was cultured at 25° C. with shaking at 200 r/min for 7 days. The ratio of the inoculation is: the seed liquid of the Cordyceps militaris mycelium is 5% of the volume of the fermentation medium.

The seed solution of the Cordyceps militaris mycelium is obtained according to the following method: inoculate the strains of the Cordyceps militaris on the seed culture medium and obtain after culturing. The solvent of the seed medium is water, and the solute and its concentration are as follows: glucose 10g/L, yeast powder 10g/L, K ₂ HPO ₄ ·3H ₂ O 0.5g/L, KH ₂ PO ₄ 0.5g/L, MgSO _{4.7H 2} O 0.5 g/L; pH 7. The culture was shaken at 25° C. at 200 r/min for 3 days.

In the step (1) of the method, after the repeated freezing and thawing of the Cordyceps militaris mycelium, before the water bath is ultrasonicated, a step of adding water to the Cordyceps militaris mycelium is also included, adding The amount of water is 40 mL of water added to the mycelium of Cordyceps militaris obtained in every 8-10 mL of the Cordyceps militaris fermentation broth. the

In step (2) of the method, the ultrasonic filtrate is obtained as follows: centrifuge the ultrasonic liquid (for example, centrifuge at 1700g for 20 minutes), take the supernatant and filter it with a 0.45 μm filter membrane, and the obtained filtrate is the ultrasonic post filtrate. the

In the step (2) of the method, the solution of the standard adenosine and/or cordycepin is an aqueous solution of the standard adenosine and/or cordycepin. the

Further, the adenosine and/or cordycepin standard substance can specifically be any of the following:

(1) Adenosine standard substance; (when the method is a method for detecting the content of adenosine in Cordyceps militaris mycelia)

(2) Cordycepin standard substance; (when the method is for detecting the content of cordycepin in Cordyceps militaris mycelia)

(3) A mixture of adenosine standard substance and cordycepin standard substance; (when the method is a method for simultaneously detecting the contents of adenosine and cordycepin in Cordyceps militaris mycelium)

More specifically, in the present invention, the adenosine standard product is a product of Sigma Company, and its catalog number is A925; the cordycepin standard product is a product of Sigma Company, and its product catalog number is C33941. the

In step (2) of the method, the "test sample obtained in step (1) and the solution of adenosine and/or cordycepin standard substance are respectively subjected to high performance liquid chromatography detection under the same conditions, According to the test result, the content of adenosine and/or cordycepin in the sample to be tested is calculated, which can be specifically:

A1) The solution of the standard adenosine and/or cordycepin with serial concentrations was detected by high-performance liquid chromatography according to the above conditions, with the concentration of the standard adenosine as the abscissa, and the standard The peak area of the product is the ordinate, and the adenosine standard curve is drawn; the sample to be tested obtained in step (1) is detected by high performance liquid chromatography according to the above conditions, and the chromatographic peak with the same retention time as the adenosine standard Bring the peak area of the adenosine standard curve into the adenosine standard curve to obtain the adenosine content in the sample to be tested; and/or

A2) The solution of the standard adenosine and/or cordycepin with serial concentrations is subjected to high-performance liquid chromatography detection according to the above conditions, the concentration of the standard cordycepin is taken as the abscissa, and the standard cordycepin The peak area of the product is the ordinate, and the standard curve of cordycepin is drawn; the sample to be tested obtained in step (1) is detected by high performance liquid chromatography according to the above conditions, and the chromatographic peak with the same retention time as the standard cordycepin The peak area is brought into the cordycepin standard curve to obtain the content of the cordycepin in the test sample. the

In A1) and A2) above, when drawing the adenosine standard curve and the cordycepin standard curve, the horizontal and vertical coordinates can be interchanged. the

In the above A1) and A2), the solution of the adenosine and/or cordycepin standard substance of the series concentration is specifically: the solvent is water, and the solute is the equal mass of the adenosine standard substance and the cordycepin The serial concentrations of the standard product, the adenosine standard product and the cordycepin standard product are all 9 μg/mL, 18 μg/mL, 27 μg/mL, 36 μg/mL, 45 μg/mL. the

In the present invention, the Cordyceps militaris is specifically the Cordyceps militaris strain CM-jd. the

Compared with the existing method, the method for detecting the content of adenosine and cordycepin in the mycelia of Cordyceps militaris provided by the present invention does not need to dry the wet mycelia cultured by liquid fermentation, and can simultaneously measure the content of adenosine and cordycepin in the mycelium. The linear range of adenosine and cordycepin is 2-50 μg/mL, the linear range of cordycepin is 3-50 μg/mL, and the detection limits of adenosine and cordycepin are 1.1 μg/mL and 2.4 μg, respectively /mL, the recoveries of the two components were 94%-100%, 99% _-103 %, respectively. This method has the advantages of saving time, energy, and fast analysis.

Description of drawings

Fig. 1 is the HPLC chromatogram of the mixture of adenosine standard substance and cordycepin standard substance. Among them, peak 1 is the standard adenosine (retention time is 5.314min), and peak 2 is the standard substance of cordycepin (retention time is 7.424min). the

Fig. 2 is the HPLC chromatogram of the sample to be tested. Among them, peak 1 is adenosine, and peak 2 is cordycepin. the

Figure 3 is the standard curve of adenosine. Wherein, the unit of concentration on the abscissa is μg/mL. the

Fig. 4 is the standard curve of cordycepin. Wherein, the unit of concentration on the abscissa is μg/mL. the

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified. the

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified. the

Instrument: High performance liquid chromatography produced by Beijing Innovation Tongheng Technology Co., Ltd., model P-3000, equipped with ultraviolet detector, data processing and acquisition rely on CXTH-3000 workstation; Milli-Q ultrapure water system (Millipore Inc, Billerica, MA, USA); Wanfen electronic balance (BP210S, Sartorius, USA); high-speed centrifuge, ultrasonic instrument;

Reagents: Methanol (HPLC grade, Fisher Scientific, USA); adenosine standard (purity ≥99%, purchased from Sigma, its catalog number is A925); cordycepin standard (purity ≥99%, purchased from Sigma , whose catalog number is C33941); 

Chromatographic column: Klomasail C18 (150mm×4.6mm, 5μm particle size), a product of AKZO NOBEL, Sweden, whose catalog number is EH04713. the

Cordyceps militaris (Cordyceps militaris) strain CM-jd: recorded in the article "Wu Fengyao. Molecular structure and biological activity of Cordyceps militaris. Jiangsu University of Science and Technology, 2011 master's thesis", which is available to the public from China Agricultural University. the

Embodiment 1, HPLC detects the establishment of the content method of adenosine and cordycepin in liquid fermentation Cordyceps militaris mycelia

1. Fermentation and cultivation of Cordyceps militaris mycelium

1. Obtaining the seed solution

Seed medium: the solvent is water, the solute and its concentration are as follows: glucose 10g/L, yeast powder 10g/L, K ₂ HPO ₄ 3H ₂ O 0.5g/L, KH ₂ PO ₄ 0.5g/L, MgSO ₄ 7H ₂ O0.5g/L; pH=7.

Inoculate the strain of Cordyceps militaris (Cordyceps militaris) strain CM-jd in a 250mL shaker flask containing 50mL of seed medium, culture at 200r/min, 25°C for 3 days to obtain seed liquid, and determine the bacterial concentration to be 1g/100mL (mycelia mass/bacteria liquid volume). the

2. Fermentation culture

Fermentation medium: the solvent is water, the solute and its concentration are as follows: sucrose 70g/L, soybean peptone 30g/L, yeast extract 30g/L, bean cake powder 15g/L, K ₂ HPO ₄ 3H ₂ O 2g/L, MgSO ₄ ·7H ₂ O 1 g/L; pH=7.

The seed liquid obtained in step 1 was inoculated into a 250mL shake flask containing 50mL fermentation medium according to the inoculum amount of 5% (volume fraction), 200r/min, 25°C for 7 days, and the fermentation liquid of Cordyceps militaris mycelium was obtained . the

2. Optimization of ultrasonic extraction conditions

1. Optimization of repeated freezing and thawing times of Cordyceps militaris mycelium

(1) Prepare the sample to be tested

Divided into 4 groups, each group took 8-10mL of Cordyceps militaris fermented liquid obtained in Step 1, put it in a 50mL centrifuge tube, centrifuged at a speed of 3500r/min (equivalent to 1700g) in a high-speed centrifuge for 15-20min, discarded Supernatant to obtain Cordyceps militaris mycelium (precipitation). Freeze the mycelium in liquid nitrogen for 20-30 minutes, then thaw it in a constant temperature water bath at 50-60°C for 10-15 minutes, and repeat the freezing and thawing several times (0, 1, 2, and 2 times for each group, respectively). 3 times; one freeze and one thaw is one freeze-thaw). Add 40mL of ultrapure water into the centrifuge tube, shake evenly, and continue ultrasonication at 40KHz in a 40°C water bath for 3h. After the ultrasonic liquid was centrifuged at 3500r/min (equivalent to 1700g) for 20min, the supernatant was taken and filtered with a 0.45μm filter membrane, and the obtained filtrate was the sample to be tested, which was used for HPLC determination. the

(2) Determination by high performance liquid chromatography

The conditions of high-performance liquid chromatography are as follows: the chromatographic column is Klomasail C18 (150mm×4.6mm, 5μm particle size); the column temperature is 25°C, the injection volume is 20μL; the mobile phase is methanol and water with a volume ratio of 15:85 The solution is mixed in proportion; the elution method is single mobile phase elution, the elution time is 15min, and the flow rate is 1mL/min. the

(3) Draw a standard curve

Weigh an appropriate amount of adenosine standard substance and cordycepin standard substance respectively, dissolve them in ultrapure water, and prepare mother liquors with a concentration of 100 μg/mL respectively; then mix appropriate amounts of the two mother liquors and dilute them with ultrapure water to prepare adenosine standard substances and cordycepin standard products with final concentrations of 9μg/mL, 18μg/mL, 27μg/mL, 36μg/mL, and 45μg/mL standard working solutions, using the HPLC conditions of step (2) to perform standard working solutions with the above series of concentrations Determination, the respective peak areas of the two standard substances were used for regression analysis on the concentration, that is, the concentration (μg/mL) of the adenosine standard (or cordycepin standard) standard working solution was used as the abscissa, and the concentration of the adenosine standard (or cordycepin standard) peak area as the ordinate, draw the standard curve. Finally, the adenosine standard curve and the cordycepin standard curve were obtained. the

(4) Data analysis

Compare the HPLC spectrum of the sample to be tested with the chromatograms of the adenosine standard and the cordycepin standard, and bring the peak area of the chromatographic peak consistent with the retention time of the adenosine standard into the adenosine standard curve obtained in step (3), Thus, the content of adenosine in the sample to be tested is calculated; the peak area of the chromatographic peak consistent with the retention time of the standard cordycepin is brought into the standard curve of cordycepin obtained in step (3), thereby calculating the content of cordycepin in the sample to be tested . the

The experiment was repeated three times, and the results were averaged. the

The results showed that in the HPLC chromatogram, the retention time of the adenosine standard was 5.314min, and the retention time of the cordycepin standard was 7.424min (Figure 1). The contents of adenosine and cordycepin in the samples to be tested were finally determined as shown in Table 1 after repeated freezing and thawing for different times. It can be seen that whether it is adenosine or cordycepin, the ultrasonic extraction effect of the freeze-thaw group is better than that of the non-freeze-thaw group (P<0.05). For adenosine, there was no significant difference in the effect of ultrasonic extraction with freeze-thaw times of 1-3 times; for cordycepin, the effect of ultrasonic extraction with freeze-thaw times of 2-3 times was better than that of freeze-thaw times of 1 time (P<0.05). the

Table 1 The contents of adenosine and cordycepin in the test samples measured by different times of repeated freezing and thawing

Note: Different lowercase letters in the same row in the table indicate significant differences at the level of P<0.05. the

2. Optimization of ultrasonic temperature of Cordyceps militaris mycelium

(1) Prepare the sample to be tested

Divided into 4 groups, each group took 8-10mL of Cordyceps militaris fermented liquid obtained in Step 1, put it in a 50mL centrifuge tube, centrifuged at a speed of 3500r/min (equivalent to 1700g) in a high-speed centrifuge for 15-20min, discarded Supernatant to obtain Cordyceps militaris mycelium (precipitation). Freeze the mycelium in liquid nitrogen for 20-30 minutes, then thaw it in a constant temperature water bath at 50-60°C for 10-15 minutes, and freeze and thaw twice in total (one freezing and one thawing are one freezing and thawing). Add 40 mL of ultrapure water into the centrifuge tube, and after shaking evenly, each group was placed in a 20°C, 30°C, 40°C, 50°C water bath for 40KHz to continue ultrasonication for 3h. After the ultrasonic liquid was centrifuged at 3500r/min (equivalent to 1700g) for 20min, the supernatant was taken and filtered with a 0.45μm filter membrane, and the obtained filtrate was the sample to be tested, which was used for HPLC determination. the

(2) Determination by high performance liquid chromatography

Same as Step 2 1(2). the

(3) Draw a standard curve

Same as Step 2 1(3). the

(4) Data analysis

Same as Step 2 1 (4). the

The experiment was repeated three times, and the results were averaged. the

The results showed that in the HPLC chromatogram, the retention time of the adenosine standard was 5.314min, and the retention time of the cordycepin standard was 7.424min (Figure 1). Table 2 shows the finally determined contents of adenosine and cordycepin in the samples to be tested at different ultrasonic temperatures. It can be seen that whether it is adenosine or cordycepin, when the ultrasonic temperature is 30-50 ℃, the ultrasonic extraction effect is better than that of the 20 ℃ group (P<0.05). For adenosine, the ultrasonic extraction effect at 40-50°C was better than that at 30°C (P<0.05); for cordycepin, there was no significant difference in the ultrasonic extraction effect at 30-50°C. the

Table 2 The content of adenosine and cordycepin in the sample to be tested measured under different ultrasonic temperatures

Note: Different lowercase letters in the same row in the table indicate significant differences at the level of P<0.05. the

3. Optimization of ultrasonic time for Cordyceps militaris mycelium

(1) Prepare the sample to be tested

Divided into 4 groups, each group took 8-10mL of Cordyceps militaris fermented liquid obtained in Step 1, put it in a 50mL centrifuge tube, centrifuged at a speed of 3500r/min (equivalent to 1700g) in a high-speed centrifuge for 15-20min, discarded Supernatant to obtain Cordyceps militaris mycelium (precipitation). Freeze the mycelium in liquid nitrogen for 20-30 minutes, then thaw it in a constant temperature water bath at 50-60°C for 10-15 minutes, and freeze and thaw twice in total (one freezing and one thawing are one freezing and thawing). Add 40mL of ultrapure water into the centrifuge tube, shake evenly, and continue ultrasonication at 40KHz in a 40°C water bath. The ultrasonication time of each group is 1h, 2h, 3h, and 4h, respectively. After the ultrasonic liquid was centrifuged at 3500r/min (equivalent to 1700g) for 20min, the supernatant was taken and filtered with a 0.45μm filter membrane, and the obtained filtrate was the sample to be tested, which was used for HPLC determination. the

(2) Determination by high performance liquid chromatography

Same as Step 2 1(2). the

(3) Draw a standard curve

Same as Step 2 1(3). the

(4) Data analysis

Same as Step 2 1 (4). the

The experiment was repeated three times, and the results were averaged. the

The results showed that in the HPLC chromatogram, the retention time of the adenosine standard was 5.314min, and the retention time of the cordycepin standard was 7.424min (Figure 1). Table 3 shows the finally determined contents of adenosine and cordycepin in the samples to be tested at different ultrasonic times. It can be seen that when the ultrasonic time is 2-4h, the ultrasonic extraction effect is better than that of other groups (P<0.05). the

Table 3 The content of adenosine and cordycepin in the test sample measured under different ultrasonic time

Note: Different lowercase letters in the same row in the table indicate significant differences at the level of P<0.05. the

Based on the optimization results of the above parameters, it can be seen that the number of repeated freezing and thawing during ultrasonic treatment is preferably 1-3 times, and 2-3 times is better. The time is preferably 2-4h. Considering the test effect and the principle of saving, it is determined that the number of repeated freezing and thawing in the follow-up test is 2 times, the ultrasonic temperature is 40°C, and the ultrasonic time is 2 hours. the

3. Determination of the recovery rate of adenosine and cordycepin under optimized conditions

(1) Prepare the sample to be tested

Take two 5mL parts of the Cordyceps militaris fermentation broth obtained in step 1, put them in 50mL centrifuge tubes (numbered 1 and 2 respectively), centrifuge at a speed of 3500r/min (equivalent to 1700g) in a high-speed centrifuge for 15-20min, discard Remove the supernatant to obtain the Cordyceps militaris mycelium (precipitate). Freeze the two copies of mycelium in liquid nitrogen for 20-30 minutes, then thaw them in a constant temperature water bath at 50-60°C for 10-15 minutes, and freeze and thaw twice in total (one freezing and one thawing are one freezing and thawing). Add 0.08mL of adenosine mother solution (concentration: 50μg/mL), 0.2mL of cordycepin mother solution (concentration: 100μg/mL) to the centrifuge tube of No. 1 sample respectively, and then add 40mL of ultrapure water; No additional adenosine and cordycepin were added to the solution, only 40mL ultrapure water was added. After the No. 1 sample tube and No. 2 sample tube were vibrated evenly, they were both placed in a water bath at 40°C at 40KHz for 2h. After the ultrasonic liquid was centrifuged at 3500r/min (equivalent to 1700g) for 20min, the supernatant was taken and filtered with a 0.45μm filter membrane, and the obtained filtrate was the sample to be tested, which was used for HPLC determination. Among them, the filtrate obtained from No. 1 sample tube was regarded as the sample after addition; the filtrate obtained from No. 2 sample tube was regarded as the sample before addition. the

(2) Determination by high performance liquid chromatography

Same as Step 2 1(2). the

(3) Draw a standard curve

Same as Step 2 1(3). the

(4) Data analysis

Referring to Step 2, 1 (4), calculate the contents of adenosine and cordycepin in the sample before and after the addition, respectively. Furthermore, the recovery rate of adenosine and cordycepin was calculated according to the following formula, recovery rate=content of adenosine or cordycepin in the sample after addition/(content of adenosine or cordycepin in the sample before addition+addition of adenosine or cordycepin amount) × 100%. the

Experiments were repeated three times. the

The results are shown in Table 4. The recoveries of adenosine and cordycepin can reach 94%-100% and 99%-103%, respectively. It can be seen that the results of the HPLC method for detecting the content of adenosine and cordycepin in the liquid fermented Cordyceps militaris mycelium provided above are accurate and reliable. the

The determination of the recovery rate of table 4 adenosine and cordycepin (unit: %)

Example 2, using the conditions optimized in Example 1 to detect the content of adenosine and cordycepin in the liquid fermentation Cordyceps militaris mycelium

1. Fermentation and cultivation of Cordyceps militaris mycelium

Same as Step 1 of Example 1. the

2. Ultrasonic extraction and HPLC detection under optimized conditions

(1) Prepare the sample to be tested

Take 8-10mL of the Cordyceps militaris fermentation liquid obtained in step 1, put it in a 50mL centrifuge tube, centrifuge at a speed of 3500r/min (equivalent to 1700g) on a high-speed centrifuge for 15-20min, discard the supernatant, and obtain Cordyceps militaris Filament (precipitation). Freeze the mycelium in liquid nitrogen for 20-30 minutes, then thaw it in a constant temperature water bath at 50-60°C for 10-15 minutes, and freeze and thaw twice in total (one freezing and one thawing are one freezing and thawing). Add 40mL of ultrapure water into the centrifuge tube, shake evenly, and continue ultrasonication at 40KHz in a 40°C water bath for 2h. After the ultrasonic liquid was centrifuged at 3500r/min (equivalent to 1700g) for 20min, the supernatant was taken and filtered with a 0.45μm filter membrane, and the obtained filtrate was the sample to be tested, which was used for HPLC determination. the

(2) Determination by high performance liquid chromatography

Same as Step 2 1 (2) of Example 1. the

(3) Draw a standard curve

Same as Step 2 1 (3) of Example 1. the

(4) Data analysis

Same as Step 2 1 (4) of Example 1. the

The experiment was repeated three times, and the results were averaged. the

The results show that:

① In the HPLC chromatogram, the retention time of the adenosine standard is 5.314min, and the retention time of the cordycepin standard is 7.424min (Figure 1). The samples to be tested have obvious chromatographic peaks at the same retention time as the adenosine standard and cordycepin standard (Figure 2). the

② As shown in Figure 3 and Figure 4, the measured linear range of adenosine is 2-50 μg/mL, and the linear range of cordycepin is 3-50 μg/mL, and the obtained peak area-concentration standard curves are respectively:

Adenosine: Y=-1.191×10 ⁵ +1.042×10 ⁵ X, r=0.998931;

Cordycepin: Y=-3.567×10 ⁵ +1.470×10 ⁵ X, r=0.997197;

③The detection limits of adenosine and cordycepin were 1.1 μg/mL and 2.4 μg/mL, respectively (the value of X when Y=0 in the standard curve). the

The average concentrations of adenosine and cordycepin in the samples finally determined were 1.65 mg/L and 4.30 mg/L, respectively (Table 5). Table 5 uses the optimized conditions of Example 1 to detect the content of adenosine and cordycepin in the liquid fermentation Cordyceps militaris mycelium

the repeat 1 repeat 2 repeat 3 average Adenosine (mg/L) 1.59 1.64 1.71 1.65 Cordycepin (mg/L) 4.26 4.34 4.29 4.30

Claims (8)

1. detect a method for the content of adenosine and/or cordycepin in cordyceps mycelium, comprise the steps:

(1) water bath sonicator is carried out by after cordyceps mycelium multigelation, using ultrasonic rear filtrate as testing sample;

(2) by the testing sample that step (1) obtains, and the solution of adenosine and/or cordycepin standard substance, carry out high performance liquid chromatography detection respectively, calculate the content of adenosine in described testing sample and/or cordycepin according to detected result.

2. method according to claim 1, is characterized in that: in step (1), and the number of times of described multigelation is 1-3 time; 1 described freeze thawing is: freezing 20-30min in liquid nitrogen, goes to the 50-60 DEG C of 10-15min that thaws.

3. method according to claim 1 and 2, is characterized in that: in step (1), and the condition of described water bath sonicator is 35-45KHz continual ultrasonic.

4., according to described method arbitrary in claim 1-3, it is characterized in that: described ultrasonic temperature is 30-50 DEG C.

5., according to described method arbitrary in claim 1-4, it is characterized in that: the described ultrasonic time is 2-4h.

6., according to described method arbitrary in claim 1-5, it is characterized in that: in step (2), the condition of described high performance liquid chromatography is as follows: chromatographic column is C18 chromatographic column; Moving phase is first alcohol and water is (10-20) according to volume ratio: the solution that the ratio of (80-90) mixes; Type of elution is single moving phase wash-out.

7., according to described method arbitrary in claim 1-6, it is characterized in that: in step (2), the flow velocity of described high performance liquid chromatography is 1mL/min, and sample size is 20 μ L, and column temperature is 25 DEG C.

8., according to described method arbitrary in claim 1-7, it is characterized in that: in step (1), described cordyceps mycelium obtains as follows: by the centrifugal 15-20min of Cordyceps militaris (L.) Link. fermented liquid 1600-1800g, abandon supernatant, obtain described cordyceps mycelium.