Method for simultaneously determining six nucleoside components in cordyceps sobolifera spore powder

Abstract

The invention discloses a method for simultaneously determining six nucleoside components in cordyceps sobolifera spore powder, and belongs to the technical field of analysis and detection. The method specifically comprises the following steps: (1) setting liquid chromatography conditions; (2) preparing a mixed reference substance stock solution; (3) preparation of a mixed reference solution and a standard curve; (4) preparing a test solution; and (5) measuring and calculating. The method disclosed by the invention has a good linear relationship in a determination range, so that the accuracy and reliability of a determination result are ensured, and a theoretical basis and an experimental method are provided for efficiently utilizing cordyceps sobolifera spore powder resources.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and more particularly to a method for simultaneously determining six nucleoside components in Cordyceps sinensis spore powder. Background Art

[0002] The asexual stage of cicada fungus was formerly known as Isaria cicadae Miq. and Paecilomyces cicadaae (Miq.) Samson. It is a fungus-insect complex formed by the parasitism of the ascomycete Cordyceps family on the mountain cicada. It is mainly distributed in East China and Southwest my country.

[0003] Cordyceps cicadae is an insect-fungus complex formed after the Cordyceps fungus infects the nymphs of Cicadaeflammata and other cicadas of the Cicadidae family. Its growth and reproduction include two stages, asexual and sexual. Asexual strains are mainly distributed in the genera Paecilomyces, Beauveria, Iosaria, and Gibellula. As a traditional Chinese medicine, cicada fungus has medicinal value similar to that of Cordyceps sinensis, with multiple effects such as improving sleep, immune regulation, anti-tumor, lowering blood pressure, lowering blood sugar, and improving kidney function. At present, cicada fungus has been artificially cultivated in factories. The 2020 National Health Commission Announcement (No. 9 of 2020) approved for the first time that artificially cultivated cicada fungus fruiting bodies can be used as new food ingredients, which has high research and development value.

[0004] Cordyceps sinensis spore powder is an asexual reproduction body (similar to "seeds") of Cordyceps sinensis. Studies have found that it is rich in nucleoside components, including uridine, adenosine, N 6 Nucleoside components such as -(2-hydroxyethyl)adenosine (HEA) play an important role in many systems of the human body. Adenosine is an important criterion for the quality evaluation of Cordyceps medicinal materials; 6 Due to its diverse pharmacological effects, 2-(2-hydroxyethyl)adenosine has also been incorporated into quality control indicators. Although Cordyceps sinensis and Cordyceps sinensis belong to the same genus, the 2015 and 2020 editions of the Pharmacopoeia of the People's Republic of China only specify a method for determining adenosine in Cordyceps sinensis, which does not fully reflect the quality of Cordyceps sinensis. The 2012 industry standard also only specifies an HPLC method for determining cordycepin and adenosine in Cordyceps sinensis.

[0005] However, the determination method of the key active ingredients (nucleoside substances) in Cordyceps sinensis spore powder has not been reported yet. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide a method for simultaneously determining six nucleoside components in Cordyceps sinensis spore powder to overcome the shortcomings of the prior art.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for simultaneously determining six nucleoside components in Cordyceps sinensis spore powder, specifically comprising the following steps:

[0009] (1) Liquid chromatography conditions

[0010] A C8 liquid chromatography column was used, and the mobile phase was water (A)-methanol (B) gradient elution;

[0011] (2) Preparation of mixed reference substance stock solution

[0012] Accurately weigh uridine, inosine, guanosine, adenosine, cordycepin and N 6 Transfer 1 mg of each 2-hydroxyethyladenosine (HEA) standard to a 10 mL volumetric flask, dissolve in water and dilute to the mark, then shake well to obtain a 0.1 mg / mL stock solution.

[0013] (3) Preparation of mixed reference solution and standard curve

[0014] Accurately pipette 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 mL of the mixed reference substance stock solution, transfer them to 10 mL volumetric flasks, add water to make up to volume and shake well to prepare a series of standard solutions of 1, 2, 5, 10, 20 and 50 μg / mL, inject the samples according to the liquid chromatography conditions of step (1), record the chromatographic peak areas of the six nucleoside substances in the mixed reference substance solution at each concentration, and draw a standard curve with mass concentration (X) as the horizontal axis and peak area (Y) as the vertical axis;

[0015] (4) Preparation of test solution

[0016] Weigh the spore powder sample of Cordyceps sinensis, add ultrapure water, shake and mix, and then ultrasonically extract. After the extraction is completed, make up the volume, mix, centrifuge, take the supernatant, filter, and perform HPLC analysis on the filtrate;

[0017] (5) Measurement and calculation

[0018] Take a sample and inject it. Perform preliminary qualitative analysis on the sample based on the retention time of the mixed standard. Combined with UV-Vis spectrometry, further verify the sample. Calculate the nucleoside content using the chromatographic peak area and standard curve. The formula for calculating the nucleoside content is:

[0019] X = C × V / m;

[0020] Where: X is the content of nucleoside substances in the sample (μg / g); C is the mass concentration of nucleoside substances converted by the standard curve (μg / mL); V is the fixed volume of the sample (mL); m is the mass of the sample (g).

[0021] Furthermore, in the above step (1), the gradient elution conditions of the mobile phase are: from 0-4 min, 5-5% (B); then from 4.0-10.0 min, 5-10% (B); 10.0-15.0 min, 10-40% (B); 15.0-17.0 min, 40-70% (B); 17.0-20.0 min, 70-85% (B); 20.0-23.0 min, 8,5→100% (B); 23.0-23.1 min, 100-5% (B); 23.1-32 min, 5-5% (B).

[0022] Furthermore, in the above step (1), the conditions for liquid chromatography analysis are: UV detection wavelength: 260 nm; column temperature: 25° C.; flow rate: 1.0 mL / min; injection volume: 20 μL; and signal acquisition time: 32 min.

[0023] Furthermore, in the above step (4), the usage ratio of the Cordyceps sinensis spore powder sample and ultrapure water is 0.5 g:20 mL.

[0024] Furthermore, in the above step (4), the ultrasonic extraction time is 70 minutes.

[0025] Furthermore, in the above step (4), the centrifugal speed is 5000 r / min and the time is 5 min.

[0026] Furthermore, in the above step (4), 0.22 μm filter membrane is used for filtration.

[0027] Furthermore, in the above step (5), the injection volume of the sample is 20 μL.

[0028] It can be seen from the above technical solution that compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] Based on the rich variety and significant therapeutic effects of nucleoside components in Cordyceps sinensis, the present invention selected six nucleoside components with high content as indicators for content determination. These components include uridine, guanosine, inosine, adenosine, cordycepin, and HEA. Not only are they abundant in Cordyceps sinensis spore powder, but they also possess unique biological activities and pharmacological effects, significantly impacting the human immune and cardiovascular systems. The present invention aims to develop a high-performance liquid chromatography method that can simultaneously, accurately, and efficiently determine the content of six nucleoside components in Cordyceps sinensis spore powder. First, a comprehensive analysis of the advantages and disadvantages of nucleoside extraction technologies was conducted. Subsequently, a series of scientific extraction condition screenings, including adjustments to the extraction method, solvent type, and ultrasonication time, were conducted to determine the optimal extraction scheme. Furthermore, a linear relationship test was conducted on the established HPLC method to ensure that the method exhibits good linearity within the measurement range, thereby ensuring the accuracy and reliability of the measurement results. This provides a theoretical basis and experimental method for the efficient utilization of Cordyceps sinensis spore powder resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Optimization of liquid chromatography gradient conditions I;

[0031] Figure 2 Optimization of liquid chromatography gradient conditions II;

[0032] Figure 3 Optimization of liquid chromatography gradient conditions III;

[0033] Figure 4 The effect of different extraction methods on the content of nucleoside substances;

[0034] Figure 5 The effect of different extraction solvents on the content of nucleoside substances;

[0035] Figure 6 The effect of different ultrasound times on the content of nucleoside substances;

[0036] Figure 7 HPLC chromatograms of the aqueous extract of Cordyceps sinensis spores and the mixed standard; Note: A is the HPLC chromatogram of the aqueous extract of Cordyceps sinensis spores, and B is the HPLC chromatogram of the mixed standard;

[0037] Figure 8 The UV spectra of uridine, inosine, guanosine and the corresponding standards in the HPLC chromatogram of the sample; Note: A, C, E are the UV spectra of uridine, inosine, and guanosine standards, respectively; B, D, and F are the UV spectra of uridine, inosine, and guanosine in the HPLC chromatogram of the sample;

[0038] Figure 9 Adenosine, cordycepin, N 6UV spectra of adenosine and its corresponding standard; Note: A, C, and E are adenosine, cordycepin, and N 6 UV spectrum of the standard product of -(2-hydroxyethyl)adenosine, B, D, and F are the HPLC chromatograms of adenosine, cordycepin, N 6 UV spectrum of -(2-hydroxyethyl)adenosine. DETAILED DESCRIPTION

[0039] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0040] Example 1

[0041] The determination method of the key active ingredients (nucleoside substances) in the spore powder of Cordyceps sinensis has not been reported yet. In the experiment of establishing the HPLC-DAD analysis method of the nucleoside components of Cordyceps sinensis spore powder, the present invention optimized the extraction process of the nucleoside components through experiments: taking 0.5g of sample, adding 20mL of ultrapure water, and ultrasonic extraction for 70min. At this time, the types of nucleoside components obtained are the most and the yield is the highest. In addition, based on the commonly used HPLC chromatography method, the chromatographic conditions were optimized, and then a set of efficient, stable and convenient HPLC chromatography analysis methods were established: chromatographic column: Agilent Eclipse Plus C8 (4.6mm×250mm, 5μm); mobile phase: gradient elution, water (A)-methanol (B); ultraviolet detection wavelength: 260nm; column temperature: 25℃; flow rate: 1.0mL / min; injection volume: 20μL; signal acquisition time is 32min. At the same time, the optimized extraction method and the established HPLC detection method were investigated in terms of stability, repeatability, linear correlation and spike recovery. The six nucleoside components had a good linear relationship within the linear range (R 2 ≥0.9998), the results showed that this method was stable and reliable, with a high recovery rate, and could be used as a detection method for the analysis of nucleoside substances in Cordyceps spore powder.

[0042] 1 Experimental methods

[0043] 1.1 Liquid chromatography conditions

[0044] An Agilent Eclipse Plus C8 column (4.6 mm × 250 mm, 5 μm) was purchased from Agilent Technologies, Inc., USA. The mobile phase consisted of a water (A)-methanol (B) gradient elution. The gradient elution conditions are shown in Table 1. UV detection wavelength: 260 nm; column temperature: 25°C; flow rate: 1.0 mL / min; injection volume: 20 μL; and signal acquisition time: 32 min.

[0045] Table 1 Gradient elution conditions

[0046]

[0047]

[0048] 1.2 Preparation of mixed reference substance stock solution

[0049] Accurately weigh 1 mg each of uridine (CAS No. 58-96-8), inosine (CAS No. 58-63-9), guanosine (CAS No. 118-00-3), adenosine (CAS No. 58-61-7), cordycepin (CAS No. 73-03-0), and HEA (CAS No. 4338-48-1) standards (purchased from Dalian Meilun Biotechnology Co., Ltd.) and transfer to a 10 mL volumetric flask. Dissolve in water and dilute to the mark. Shake well to obtain a 0.1 mg / mL stock solution. This stock solution must be stored in a refrigerator at 4°C and is valid for 1 month.

[0050] 1.3 Preparation of mixed reference solution and standard curve

[0051] Accurately pipette 0.1, 0.2, 0.5, 1.0, 2.0, and 5.0 mL of the mixed reference stock solution into 10 mL volumetric flasks, dilute to volume with water, and shake well to prepare a series of standard solutions at 1, 2, 5, 10, 20, and 50 μg / mL. Inject the samples according to the chromatographic conditions described in "1.1" and record the peak areas of the six nucleoside compounds in the mixed reference solution at each concentration. Plot a standard curve with mass concentration (X) as the horizontal axis and peak area (Y) as the vertical axis.

[0052] 1.4 Preparation of test solution

[0053] Accurately weigh 0.5 g of Cordyceps sinensis spore powder (purchased from Hangzhou Chansheng Pharmaceutical Co., Ltd.) and add 20 mL of ultrapure water. After vortexing and mixing, extract the sample using a JY96-IIN ultrasonic disruptor (Ningbo Xinzhi Biotechnology Co., Ltd.) for 70 min. After extraction, adjust the volume, mix thoroughly, and centrifuge at 5000 rpm for 5 min. The supernatant was filtered through a 0.22 μm filter membrane, transferred to a vial, and stored at 4°C for HPLC analysis using a Thermo Ultimate 3000 UHPLC (Thermo Fisher Scientific, USA).

[0054] 1.5 Measurement and calculation

[0055] Take 20 μL of sample and inject it. Perform preliminary qualitative analysis of the sample based on the retention time of the mixed standard and further verify it with the UV-Vis spectrum. Calculate the nucleoside content based on the chromatographic peak area and the standard curve. If the sample response value exceeds the range of the standard curve, it needs to be diluted and re-measured. The formula for calculating the nucleoside content is:

[0056] X = C × V / m;

[0057] Where: X is the content of nucleoside substances in the sample (μg / g); C is the mass concentration of nucleoside substances converted by the standard curve (μg / mL); V is the fixed volume of the sample (mL); m is the mass of the sample (g).

[0058] 2 Experimental condition selection and optimization

[0059] 2.1 Optimization of chromatographic conditions

[0060] (1) Selection of gradient elution optimization program

[0061] 1) Optimization Program I

[0062] Since the isocratic elution method is difficult to effectively separate the nucleoside components in the ideal capacity factor range when separating the spore powder of Cordyceps sinensis, and the chromatographic peaks of some components to be tested overlap, gradient elution is adopted. After a series of experiments, the initial gradient elution program shown in Table 2 was determined, and the chromatogram is shown in Figure 1 .

[0063] Gradient elution: from 0-4 min, 2→2% (B); then from 4.0-10.0 min, 2→10% (B), 10.0-15.0 min, 10→40% (B); 15.0-16.0 min, 40→100% (B); 16.0-20.0 min, 100→100% (B); 20.0-20.5 min, 100→2% (B); 20.5-25 min, 2→2% (B).

[0064] Table 2 Optimization of liquid chromatography gradient conditions I

[0065]

[0066]

[0067] Depend on Figure 1 As can be seen, the chromatographic peaks are dense within 10-15 minutes, indicating that the elution strength of the mobile phase is set too high during this period. The chromatographic peaks are also dense within 15-20 minutes, and the baseline drift is severe, indicating that the mobile phase changes are too large and the gradient steepness needs to be reduced. The column equilibration time should also be appropriately extended.

[0068] 2) Optimization Program II

[0069] The elution procedure is shown in Table 3 and the chromatogram is shown in Figure 2 .

[0070] Table 3 Optimization of liquid chromatography gradient conditions II

[0071] Time (min) water(%) Methanol (%) 0 95 5 4 95 5 10 90 10 15 60 40 20 25 75 23 0 100 23.1 95 5 28 95 5

[0072] Depend on Figure 2 It can be seen that when the gradient steepness is reduced from 15 to 20 minutes, the peaks are moderately separated, but there is asymmetry in the chromatographic peaks. The change amplitude of the eluent in this time period is further reduced.

[0073] 3) Optimization Program III

[0074] The elution procedure is shown in Table 4 and the chromatogram is shown in Figure 3 .

[0075] Table 4 Optimization of liquid chromatography gradient conditions III

[0076] Time (min) water(%) Methanol (%) 0 95 5 4 95 5 10 90 10 15 60 40 17 30 70 20 15 85 23 0 100 23.1 95 5 32 95 5

[0077] Depend on Figure 3 As can be seen, when the gradient steepness is further reduced for 15-20 minutes and the organic phase ratio is appropriately adjusted, the elution time reaches 32 minutes, resulting in a stable baseline and good peak separation. Under the conditions of Gradient Elution III, the analytes achieve excellent baseline separation, with moderate retention times. Not only are the chromatographic peak shapes good, but chromatographic reproducibility is also good, and no tailing of the analyte peaks is observed. Therefore, the chromatographic conditions of Optimization Procedure III were selected as the final chromatographic conditions.

[0078] 2.2 Selection of test sample preparation conditions

[0079] (1) Comparison of extraction methods

[0080] The purpose of this experiment was to explore the extraction efficiency of nucleoside substances in Cordyceps sinensis spore powder. By comparing two different extraction methods, ultrasonic extraction and traditional water boiling, the effect of extraction method on the extraction rate of nucleoside substances was determined.

[0081] 1) Ultrasonic extraction method

[0082] Prepare the test solution in the same way as in 1.4.

[0083] 2) Water boiling extraction method

[0084] Weigh 10 g of spore powder and add 1 L of distilled water. Boil for 30 minutes and stir until completely dissolved. Top up with distilled water, cool, and centrifuge at 5000 rpm for 5 minutes. Concentrate the supernatant on a rotary evaporator, filter through a 0.22 μm filter, and transfer the filtrate to a vial for HPLC analysis.

[0085] Depend on Figure 4 It can be seen that the ultrasonic extraction method has higher uridine, guanosine, and adenosine contents than the traditional water boiling method, and the ultrasonic extraction method can obtain more types of nucleoside substances than the traditional water boiling method. Therefore, the ultrasonic extraction method was selected in this experiment.

[0086] (2) Selection of extraction solvent

[0087] Based on the structure and physicochemical properties of nucleoside substances, this experiment used water, 60% ethanol, 75% ethanol, 10% methanol, 50% methanol and anhydrous methanol as extraction media to evaluate the effect of different solvents on the extraction rate of nucleoside substances. Accurately weigh 0.5g of sample, add the above extraction solvents respectively, and perform the determination according to the steps described in "1.4" and "1.5". The results are shown in the figure. Figure 5 .

[0088] Depend on Figure 5 It can be seen that among the six different extraction solvents, water has the best extraction effect. The nucleoside substances extracted by it not only have the most types but also the highest measured values. Therefore, this experiment finally chose water as the ideal solvent for extracting nucleoside substances from Cordyceps sinensis spore powder.

[0089] (3) Selection of extraction time

[0090] Accurately weigh 0.5 g of sample, add 20 mL of ultrapure water, vortex to mix, and analyze and determine according to the above "1.4" and "1.5" steps except for ultrasonic treatment time of 30 min, 50 min, 70 min, and 90 min respectively.

[0091] Depend on Figure 6 As can be seen, the levels of the five nucleosides, except HEA, were highest when the ultrasound duration was 70 minutes. The HEA content was similar to that of the other treatment time groups. Therefore, a 70-minute ultrasound duration was appropriate for this experiment.

[0092] 3 Results and Analysis

[0093] 3.1 Sample determination

[0094] The mixed reference substance and different Cordyceps spore powder samples were tested according to the chromatographic conditions of "1.1". The HPLC chromatograms of the Cordyceps sinensis spore water extract and the mixed reference substance are shown in Figure 1. Figure 7 Under these experimental conditions, the six nucleoside components in the sample achieved good chromatographic separation with good peak shapes, and all peaks were eluted within 32 minutes.

[0095] In the HPLC fingerprint of the spore powder water extract, six characteristic chromatographic peaks 9#, 11#, 12#, 16#, 17# and 18# were observed. The retention time and UV spectral characteristics of the peaks to be tested in the reference substance and the sample were investigated through the control experiment ( Figure 8-9 ), preliminary characterization was performed by comparing the retention time with that of a mixed standard solution, and further characterization was performed by comparing the UV-Vis spectra. The six characteristic chromatographic peaks were identified as uridine, inosine, guanosine, adenosine, cordycepin, and HEA. The optical information provided by their respective UV spectra was generally consistent with the spectral characteristics of the known standard chromatographic peaks.

[0096] 3.2 Standard curve and linear relationship

[0097] For the nucleoside standard products, linear regression analysis was performed using HPLC analysis, with the chromatographic peak area as the ordinate and the concentration of the nucleoside standard solution as the abscissa. The resulting regression equation is shown in Table 5.

[0098] Table 5 Regression analysis of 6 nucleoside components

[0099] Element Regression equation Correlation coefficient Uridine Y=1.3257X+0.0287 <![CDATA[R 2 =0.9999]]> Inosine Y=0.6387X-0.0252 <![CDATA[R 2 =1.0000]]> Guanosine Y=0.5456X-0.0198 <![CDATA[R 2 =1.0000]]> adenosine Y=0.3184X-0.0107 <![CDATA[R 2 =0.9999]]> Cordycepin Y=0.3757X-0.0062 <![CDATA[R 2 =0.9999]]> <![CDATA[N 6 -(2-Hydroxyethyl)adenosine]]> Y=1.3269X+0.0961 <![CDATA[R 2 =0.9998]]>

[0100] As shown in Table 5, all correlation coefficients are greater than 0.9998, indicating that within the linear range, there is a good linear relationship between the peak area of ​​the standard and its concentration.

[0101] 3.3 Methodological Investigation

[0102] 3.3.1 Precision investigation

[0103] The Cordyceps sinensis spore powder sample solution prepared in step "1.4" was injected six times continuously under the chromatographic conditions in step "1.1". The peak areas of the nucleoside substances were recorded and the precision was calculated. The results are shown in Table 6.

[0104] Table 6 Precision experiment

[0105]

[0106] As shown in Table 6, the RSD values ​​were all less than 2.5% (n=6), indicating that the instrument had good precision.

[0107] 3.3.2 Repeatability study

[0108] Six parallel samples of Cordyceps sinensis spore powder were prepared according to step 1.4 and injected under the chromatographic conditions described in step 1.1 to investigate the repeatability of the method. Peak areas were recorded. The results are shown in Table 7.

[0109] Table 7 Repeatability experiment

[0110]

[0111] As shown in Table 7, the RSD values ​​were all less than 2% (n=6), indicating that the method had good repeatability.

[0112] 3.3.3 Stability investigation

[0113] Accurately weigh 0.5 g of the sample and prepare the test solution according to "1.4". Samples were injected and analyzed every few hours within 24 hours of preparation under the chromatographic conditions described in "1.1" to investigate the stability of the nucleoside content in the Cordyceps sinensis spore powder sample. The results are shown in Table 8.

[0114] Table 8 Stability test

[0115]

[0116] As shown in Table 8, the RSD values ​​were all less than 4.2%, indicating that the test solutions were stable within 24 h.

[0117] 3.3.4 Spike recovery experiment

[0118] Weigh 6 portions of 0.5g Cordyceps sinensis spore powder, add appropriate amounts of standard, prepare sample solutions according to "1.4", and assay according to "1.1" to calculate the recovery. The RSD values ​​for the six nucleoside compounds were 0.47%, 1.73%, 0.59%, 0.78%, 1.72%, and 1.16%, respectively. The results are shown in Table 9.

[0119] Table 9 Spiked recovery experimental results

[0120]

[0121]

[0122] As shown in Table 9, the chromatographic conditions and extraction method have good recovery rates and are suitable for the determination of nucleoside content in Cordyceps spore powder.

[0123] 3.3.5 Limit of Quantitation and Limit of Detection

[0124] The mixed reference solution of "1.3" was gradually diluted with water. The detection limit and quantification limit of the six nucleoside substances were obtained by taking the target mass concentration corresponding to 3 times the signal-to-noise ratio as the detection limit and the target mass concentration corresponding to 10 times the signal-to-noise ratio as the quantification limit. The results showed that the detection limits and quantification limits of the six nucleoside substances were 3.3×10 -3 μg / mL, 1.11×10 -2 μg / mL, 8.4×10 -3 μg / mL, 1.18×10 -2 μg / mL, 8.5×10 -3 μg / mL, 2.1×10 -3 μg / mL. Their LOQs were 1.1×10 -2 μg / mL, 3.69×10 -2 μg / mL, 2.8×10 -2 μg / mL, 3.92×10 -2 μg / mL, 2.82×10 -2 μg / mL, 7.1×10 -3 μg / mL.

[0125] In summary, nucleosides are composed of bases and sugars linked by covalent bonds and exhibit a wide range of pharmacological activities. Cicada fungus contains a variety of nucleosides, and the nucleosides in Cordyceps herbs vary depending on their origin, climate, and host species.

[0126] Cordyceps sinensis is a medicinal and edible fungus with health benefits. It contains multiple nucleoside components. It is difficult to control the quality of Cordyceps products with a single adenosine component. Although researchers have used HPLC to determine the nucleoside substances in some Cordyceps, most of them use 1-3 nucleoside components as quality control indicators for evaluation. The fingerprint of traditional Chinese medicine can generate a spectrum representing the characteristic common peaks of the traditional Chinese medicine, comprehensively reflecting its chemical properties. It can be used to evaluate the quality of traditional Chinese medicine, identify authenticity and the consistency of production characteristics. By measuring the area and ratio of the main characteristic peaks, the sample quality can be effectively controlled. The present invention uses HPLC-DAD combined technology to establish a chromatographic method for the simultaneous determination of six nucleoside components in several Cordyceps spore powders, and establishes traditional Chinese medicine fingerprints for different Cordyceps spore powders from different culture media and Cordyceps B spore powder stored under different storage conditions; and using nematodes as model organisms, the effect of the aqueous extract of Cordyceps B spore powder on the oxidative stress of nematodes is comprehensively evaluated based on the health status and stress capacity of the nematodes.

[0127] The main results are as follows: Based on analysis of sample preparation conditions, ultrasonic extraction was selected as the optimal pretreatment method for determining the content of nucleoside compounds in Cordyceps spore powder, using water as the extraction solvent and a treatment time of 70 minutes. Based on the properties of the nucleoside compounds, appropriate extraction and separation methods were employed. By optimizing chromatographic conditions, a method for the simultaneous determination of six nucleoside compounds in Cordyceps sinensis spore powder was established using HPLC-DAD technology. Furthermore, the optimized extraction method and the established high-performance liquid chromatography (HPLC) detection technique were systematically evaluated for stability, reproducibility, linear response range, and spike recovery efficiency. This chromatographic method yielded symmetrical and clear peaks, with ideal separation results, making it suitable for use as a detection method for the analysis of nucleoside compounds in Cordyceps sinensis spore powder.

[0128] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for simultaneously determining six nucleoside components in Cordyceps sinensis spore powder, characterized in that: The specific steps include: (1) Liquid chromatography conditions A C8 liquid chromatography column was used, and the mobile phase was water (A)-methanol (B) gradient elution; (2) Preparation of mixed reference substance stock solution Accurately weigh uridine, inosine, guanosine, adenosine, cordycepin and N 6 Transfer 1 mg of each 2-hydroxyethyladenosine standard to a 10 mL volumetric flask, dissolve in water and dilute to the mark, then shake well to obtain a 0.1 mg / mL stock solution. (3) Preparation of mixed reference solution and standard curve Accurately pipette 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 mL of the mixed reference substance stock solution, transfer them to 10 mL volumetric flasks, add water to make up to volume and shake well to prepare a series of standard solutions of 1, 2, 5, 10, 20 and 50 μg / mL, inject the samples according to the liquid chromatography conditions of step (1), record the chromatographic peak areas of the six nucleoside substances in the mixed reference substance solution at each concentration, and draw a standard curve with mass concentration (X) as the horizontal axis and peak area (Y) as the vertical axis; (4) Preparation of test solution Weigh the spore powder sample of Cordyceps sinensis, add ultrapure water, shake and mix, and then ultrasonically extract. After the extraction is completed, make up the volume, mix, centrifuge, take the supernatant, filter, and perform HPLC analysis on the filtrate; (5) Measurement and calculation Take a sample and inject it. Perform preliminary qualitative analysis on the sample based on the retention time of the mixed standard. Combined with UV-Vis spectrometry, further verify the sample. Calculate the nucleoside content using the chromatographic peak area and standard curve. The formula for calculating the nucleoside content is: X = C × V / m; Where: X is the content of nucleoside substances in the sample (μg / g); C is the mass concentration of nucleoside substances converted by the standard curve (μg / mL); V is the fixed volume of the sample (mL); m is the mass of the sample (g).

2. The method for simultaneously determining six nucleoside components in Cordyceps sinensis spore powder according to claim 1, characterized in that: In step (1), the gradient elution conditions of the mobile phase are: from 0-4 min, 5-5% (B); then from 4.0-10.0 min, 5-10% (B); 10.0-15.0min, 10-40% (B); 15.0-17.0 min, 40-70% (B); 17.0-20.0min, 70-85% (B); 20.0-23.0min, 8,5→100%(B); 23.0-23.1min, 100-5%(B); 23.1-32min, 5-5%(B).

3. The method for simultaneously determining six nucleoside components in Cordyceps sinensis spore powder according to claim 1, characterized in that: In step (1), the conditions for the liquid chromatography analysis are: ultraviolet detection wavelength: 260 nm; column temperature: 25° C.; flow rate: 1.0 mL / min; injection volume: 20 μL; and signal acquisition time: 32 min.

4. The method for simultaneously determining six nucleoside components in Cordyceps sinensis spore powder according to claim 1, characterized in that: In step (4), the usage ratio of the Cordyceps sinensis spore powder sample and ultrapure water is 0.5g:20mL.

5. The method for simultaneously determining six nucleoside components in Cordyceps sinensis spore powder according to claim 1, characterized in that: In step (4), the ultrasonic extraction time is 70 minutes.

6. The method for simultaneously determining six nucleoside components in Cordyceps sinensis spore powder according to claim 1, characterized in that: In step (4), the centrifugal speed is 5000 r / min and the time is 5 min.

7. The method for simultaneously determining six nucleoside components in Cordyceps sinensis spore powder according to claim 1, characterized in that: In step (4), the filtration uses a 0.22 μm filter membrane.

8. The method for simultaneously determining six nucleoside components in Cordyceps sinensis spore powder according to claim 1, characterized in that: In step (5), the injection volume of the sample is 20 μL.

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