CN102928526A - Method for analyzing content of adenosine and cordycepin in cordyceps militaris by virtue of high performance liquid chromatography (HPLC) - Google Patents
Method for analyzing content of adenosine and cordycepin in cordyceps militaris by virtue of high performance liquid chromatography (HPLC) Download PDFInfo
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Abstract
The invention discloses a method for analyzing the content of adenosine and cordycepin in the sporocarp of cordyceps militaris through artificial culture by virtue of high performance liquid chromatography (HPLC). The method comprises the following specific steps: 1, weighing the dried sporocarp powder of cordyceps militaris in a 50ml centrifuge tube with a plug, adding water, ultrasonically extracting the centrifuge tube in water bath, then centrifuging and taking a supernate as a test solution; 2, weighing a certain amount of standard substances of adenosine and cordycepin, adding water and dissolving to prepare a mixed solution as a mixed standard solution; 3, performing content measuring, namely taking the mixed standard solution and test solution respectively, filtering the solutions through a 0.45um aqueous phase millipore filter and injecting the filtered solutions into a small bottle, and measuring the chromatographic peak of the cordyceps militaris sporocarp test solution and the adenosine cordycepin standard solution with the same chromatographic retention time. The invention provides a new, simple and convenient method for evaluating the quality of the cordyceps militaris sporocarp.
Description
Technical field
The present invention relates to the analytical chemistry field, particularly the method for adenosine and cordycepin content in a kind of high performance liquid chromatography (HPLC) method analysis fruiting bodies of cordyceps militaris.
Background technology
Cordyceps militaris (having another name called northern Chinese caterpillar fungus, northern Chinese caterpillar Fungus) is the dry stroma of Clavicipitaceae Cordyceps sinensis fungus pupa grass bacterium (Cordyceps militaris (L.) Link), be the distinctive famous and precious medicinal fungi of China, have the multiple pharmacologically actives such as expansion organ, calmness, anti-arrhythmia, hypotensive, resisting pathogenic microbes, anti-malignant tumor.The gradient elution such as adenosine, cordycepin is used as the main reference index of Chinese caterpillar fungus quality.Adenosine (C
10H
13N
5O
4, (a) that structural formula is as follows) and the full name adenosine, be the fundamental structural unit of nucleic acid, it has the function that participates in body metabolism and physiology adjusting.Cordycepin (C
10H
13N
5O
3, (b) that structural formula is as follows) and claim again cordycepin, 3 ＇-desoxyadenossine, have the effects such as antibiotic, anti-inflammatory, anti-oxidant, antitumor, balance the body endocrine.
The assay of adenosine and cordycepin is the prerequisite of worm grass product quality control and study metabolic pathways in the fruiting bodies of cordyceps militaris.At present, the assay of adenosine and cordycepin is the most general with high performance liquid chromatography in the Cordyceps militaris, but the mixed system that adopts phosphate solution and organic phase is as mobile phase more, and uses for a long time the relative liquid phase systems injury of salt larger.The people such as Liu Xiaofang (Liu Xiaofang, Xue Changhu, Chang Yaoguang. the content of adenosine and cordycepin [J] in the high effective liquid chromatography for measuring Cordyceps militaris. Food Science, 2010,31 (16): 172-175) proposed the method for adenosine and cordycepin content in a kind of new mensuration Cordyceps militaris, its mobile phase is comprised of citric acid, acetic acid, triethylamine mixed solution and acetonitrile, and the composition of mobile phase and preparation process are comparatively complicated.The people such as Li Xiangling (Li Xiangling, Hu Jingsong, oldly make red .HPLC and measure cordycepin and adenosine content [J] in artificial Chinese caterpillar fungus and the nutrient culture media thereof. Journal of Natural Science of Hunan Normal University, 2010,33 (2): 107-110), the people (Mao Xinliang such as Mao Xinliang, Zheng Guodong, Zhang Chen, Deng. reversed-phased high performace liquid chromatographic is measured the research [J] of adenosine in the Cordyceps sinensis, cordycepin, flesh liver content method simultaneously. Central-South pharmacy, 2009,7 (12): though 895-897) with the first alcohol and water as mobile phase, adopt the mode of gradient elution, process is loaded down with trivial details.In addition, if adopt the extraction conditions of adenosine in " health food check and assessment technique standard " version in 2003 that Cordyceps militaris fruiting body is extracted, then extraction ratio is on the low side.
Therefore at present, the assay of adenosine and cordycepin does not also have unified national standard method in the Cordyceps militaris, establishes the content that a disposable sample introduction of easy reliable method analyzes adenosine and Cordyceps militaris simultaneously and has realistic meaning.
Summary of the invention
The present invention is take tame fruiting bodies of cordyceps militaris as material, water is extract, adopting ultrapure water and methyl alcohol is the mobile phase isocratic elution, set up a kind of single injected sampling and can measure simultaneously the high efficiency liquid phase chromatographic analysis method of adenosine and cordycepin content in the fruiting bodies of cordyceps militaris, and extraction conditions has been carried out more comprehensive optimization.The assay method of adenosine and cordycepin content in use high performance liquid chromatography provided by the invention (HPLC) the method mensuration fruiting bodies of cordyceps militaris, have and extract and analytic process is easy fast, the recovery is high, the characteristics of good reproducibility, can be fast, adenosine and cordycepin content in the Accurate Determining fruiting bodies of cordyceps militaris, effectively measure and control Tissue Culture of Cordyceps militaris fructification product quality.
Technical scheme of the present invention is as follows:
The method of adenosine and cordycepin content comprises the steps: in a kind of high-efficient liquid phase chromatogram technique analysis fruiting bodies of cordyceps militaris
(1) adenosine and cordycepin mixed standard solution preparation: take by weighing the adenosine and the cordycepin standard items that are dried to constant weight and place container, water dissolving and constant volume add adenosine and cordycepin mixed standard solution that ultrapure water is diluted to described standard reserving solution some variable concentrations as standard reserving solution;
Fruiting bodies of cordyceps militaris test solution preparation: fruiting bodies of cordyceps militaris is dried, through the comminutor grinding and sieving, take by weighing the fruiting bodies of cordyceps militaris powdered sample that is dried to constant weight, add extract and extract, separate to get supernatant after extracting end, be the fruiting bodies of cordyceps militaris test solution;
(2) measure: draw respectively adenosine and cordycepin mixed standard solution and fruiting bodies of cordyceps militaris test solution, inject the sample introduction bottle after filtering, measure the fruiting bodies of cordyceps militaris test solution and should present the chromatographic peak identical with cordycepin mixed standard solution chromatographic retention with adenosine.
Preferably, in the described step (1), the preparation method of described adenosine and cordycepin mixed standard solution is: accurately take by weighing adenosine and each 4mg of cordycepin standard items of being dried to constant weight, place 50 ml volumetric flasks, as standard reserving solution, concentration is designated as 80 μ g/ml with the water-soluble solution of 50ml and constant volume;
Described standard reserving solution is diluted to adenosine and the cordycepin mixed standard solution of 2 μ g/ml, 5 μ g/ml, 10 μ g/ml, 20 μ g/ml, 40 μ g/ml, 80 μ g/ml with ultrapure water.
Preferably, described accurately taking by weighing as being accurate to 0.0001 g.
Preferably, in the described step (1), the fruiting bodies of cordyceps militaris test solution is prepared as: fruiting bodies of cordyceps militaris is placed 40 ℃ of drying baker oven dry, after pulverizing, crosses by comminutor 200 mesh sieves, accurately take by weighing be dried to constant weight sample 0.50 g in 50 ml centrifuge tubes, add the ultrasonic extraction of water-bath behind the 25 ml extracts, after extract finishing centrifuge tube placed 20 ℃, centrifugal 10 min of hydro-extractor of 5000 r/min, draw supernatant 1 ml, behind 0.45 μ m water filtering with microporous membrane, directly inject sample injection bottle for analysis.
Preferably, in the preparation of described step (1) fruiting bodies of cordyceps militaris test solution, described extract is pure water.
Preferably, in described step (1) the fruiting bodies of cordyceps militaris test solution preparation, describedly be extracted as extraction once, extraction time is 30-40 minute, and extracting temperature is 30 ℃.
Preferably, the ultrasonic power of the ultrasonic extraction of described water-bath is 700W.
Preferably, in the described step (2), high-efficient liquid phase chromatogram condition is:
Chromatographic column is XBridgeTM C18 (5 μ m, 4.6 * 250 mm);
Mobile phase is methyl alcohol: water=16:84(v:v);
Flow velocity: 1.0 ml/min;
Sample size: 10 μ L, auto injection;
Column temperature: 30 ℃;
Detect wavelength: 254 nm.
Preferably, the water in the described mobile phase is ultrapure water.
Preferably, described chromatographic determination process adopts isocratic elution.
Compared with prior art, beneficial effect of the present invention is as follows:
The method that a kind of high performance liquid chromatography single injected sampling is measured adenosine and cordycepin in the fruiting bodies of cordyceps militaris has simultaneously been established in this experiment, and the sample extraction condition has been carried out more comprehensive optimization.
The method mainly contains following advantage:
1) with water as extract, simply pollution-free, the fruiting bodies of cordyceps militaris sample pre-treatments is simple, and is ultrasonic through water-bath, disposable extraction gets final product;
2) high, the good stability of the recovery, the recovery of standard addition of adenosine and cordycepin is respectively 95.78 %, 94.35 %, and the RSD of replication is respectively 3.311 %, 3.313 % (n=6);
3) compare with the analytical approach of having reported, simplified the composition of mobile phase, take the first alcohol and water as mobile phase, simply need not complicated process for preparation, especially avoided the use of buffer salt to the damage of instrument, adopt isocratic elution, make analytic process extremely easy.
The inventive method provides reference frame for the analysis of adenosine in the fruiting bodies of cordyceps militaris from now on and cordycepin content.
Certainly, implement arbitrary product of the present invention and might not need to reach simultaneously above-described all advantages.
Description of drawings
Figure 1A, Figure 1B are respectively the typical curve of adenosine and cordycepin in the embodiment of the invention 1;
Fig. 2 be in the embodiment of the invention 1 extract concentration of alcohol on recording the impact of adenosine and cordycepin content;
The chromatogram that Fig. 3 measures a sample for using the present invention.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be appreciated that these embodiment only are used for explanation the present invention, and are not intended to limit the scope of the invention.In actual applications improvement and the adjustment made according to the present invention of those skilled in the art still belongs to protection scope of the present invention.
The method of adenosine and cordycepin content comprises the steps: in a kind of high-efficient liquid phase chromatogram technique analysis fruiting bodies of cordyceps militaris of the present invention
(1) adenosine and cordycepin mixed standard solution preparation: accurately take by weighing the adenosine and the cordycepin standard items that are dried to constant weight and place container, water dissolving and constant volume add adenosine and cordycepin mixed standard solution that ultrapure water is diluted to described standard reserving solution some variable concentrations as standard reserving solution; Wherein more excellent scheme is: accurately take by weighing adenosine and each 4mg of cordycepin standard items of being dried to constant weight, place 50 ml volumetric flasks, use the water-soluble solution of 50ml and constant volume as standard reserving solution, concentration is designated as 80 μ g/ml; Described standard reserving solution is diluted to adenosine and the cordycepin mixed standard solution of 2 μ g/ml, 5 μ g/ml, 10 μ g/ml, 20 μ g/ml, 40 μ g/ml, 80 μ g/ml with ultrapure water.Preferably, taking by weighing of standard items is accurate to 0.0001 g.Herein to mixed standard solution prepare Plays product quality, the concentration of volume, the mixed standard solution that obtains of configuration that takes by weighing precision, container and volume thereof, water is only for for example, the scheme that any those skilled in the art obtain the simple replacement of above-mentioned condition is all in protection scope of the present invention.
Fruiting bodies of cordyceps militaris test solution preparation: fruiting bodies of cordyceps militaris is dried, through the comminutor grinding and sieving, accurately take by weighing the fruiting bodies of cordyceps militaris powdered sample that is dried to constant weight, add extract and extract, separate to get supernatant after extracting end, be the fruiting bodies of cordyceps militaris test solution; Wherein more excellent scheme is: fruiting bodies of cordyceps militaris is placed 40 ℃ of drying baker oven dry, after pulverizing, crosses by comminutor 200 mesh sieves, accurately take by weighing be dried to constant weight sample 0.50 g in 50 ml centrifuge tubes, add the ultrasonic extraction of water-bath behind the 25 ml extracts, after extracting end centrifuge tube is placed 20 ℃, centrifugal 10 min in the hydro-extractor of 5000 r/min, draw supernatant 1 ml, behind 0.45 μ m water filtering with microporous membrane, directly inject sample injection bottle for analysis, better, extract is pure water, extracts once, extraction time is 30-40 minute, better be 40 minutes, extracting temperature is 30 ℃, and the ultrasonic power of the ultrasonic extraction of water-bath is 700W.Container volume, powdered sample quality, extracting liquid volume, separate mode and condition should not be construed as limitation of the present invention only for for example in above-mentioned bake out temperature, mistake grit number and the fruiting bodies of cordyceps militaris powdered sample preparation process.The scheme that any those skilled in the art obtain the simple replacement of above-mentioned condition is all in protection scope of the present invention.
In the methods of the invention, the preparation of the preparation of adenosine and cordycepin mixed standard solution and fruiting bodies of cordyceps militaris test solution in no particular order, can carry out first the preparation of adenosine and cordycepin mixed standard solution and carry out again the preparation of fruiting bodies of cordyceps militaris test solution, perhaps carry out first the preparation of fruiting bodies of cordyceps militaris test solution and carry out the preparation of adenosine and cordycepin mixed standard solution again, perhaps the preparation of the preparation of adenosine and cordycepin mixed standard solution and fruiting bodies of cordyceps militaris test solution is carried out simultaneously.
(2) measure: precision is drawn adenosine and cordycepin mixed standard solution and fruiting bodies of cordyceps militaris test solution respectively, inject the sample introduction bottle after filtering, measure the fruiting bodies of cordyceps militaris test solution and should present the chromatographic peak identical with Cordycepin mixed standard solution chromatographic retention.Wherein, high-efficient liquid phase chromatogram condition is:
Chromatographic column is XBridgeTM C18 (5 μ m, 4.6 * 250 mm);
Mobile phase is methyl alcohol: water=16:84(v:v);
Flow velocity: 1.0 ml/min;
Sample size: 10 μ L, auto injection;
Column temperature: 30 ℃;
Detect wavelength: 254 nm.
In preferred version of the present invention, the wet concentration ultrapure water in the mobile phase, the chromatographic determination process adopts the isocratic elution method.
Embodiment 1
The method of adenosine and cordycepin content in the high performance liquid chromatography that present embodiment provides (HPLC) the method mensuration fruiting bodies of cordyceps militaris comprises following several part:
1, instrument and reagent
Instrument: Waters e2695 type high performance liquid chromatograph; Waters 2489 ultraviolet-visible detectors; Millinium workstation and empower3 data handling system, more than all available from U.S. Waters company; KH-700E type ultrasonic cleaner; The preparation of Milli-Q ultrapure water and distribution instrument;
Test sample: fruiting bodies of cordyceps militaris, cultivated with Chinese caterpillar fungus seminar of biological institute by Shanghai Communications University's agricultural;
Reagent: adenosine standard items, purity〉98%, source, Shanghai leaf bio tech ltd provides; Cordycepin standard items, purity〉98%, upper Hiroad standing grain Pharmaceutical Technology Co., Ltd provides; Absolute methanol, chromatographically pure, JTBaker chemical products trade Co., Ltd provides; Absolute ethyl alcohol, analyze pure, in nine Science and Technology Ltd.s provide; Ultrapure water is through the preparation of Milli-Q ultrapure water and the preparation of distribution instrument; It is pure that other reagent is commercially available analysis.
2, adenosine and cordycepin content are measured
2.1, chromatographic condition
Chromatographic column: the XBridgeTM C18 (5 μ m, 4.6 x, 250 mm) that is provided by waters company;
Mobile phase: methyl alcohol: the volume ratio of ultrapure water is 16:84;
Detect wavelength: 254nm
Flow velocity: 1.0ml/min;
Column temperature: 30 ℃
Sample size: 10 L, auto injection.The chromatographic determination process adopts the isocratic elution method.
2.2、
A. the preparation of reference substance mixed standard solution
Accurately take by weighing adenosine and each 4mg of cordycepin standard items (being accurate to 0.0001g) of being dried to constant weight and be added in the 50 ml volumetric flasks, water dissolving and constant volume are as standard reserving solution, and concentration is designated as 80 μ g/ml; Above-mentioned standard reserving solution is diluted to adenosine and the cordycepin mixed standard solution of 2 μ g/ml, 5 μ g/ml, 10 μ g/ml, 20 μ g/ml, 40 μ g/ml, 80 μ g/ml with ultrapure water.
B. the preparation of fruiting bodies of cordyceps militaris need testing solution
Fruiting bodies of cordyceps militaris is placed 40 ℃ of drying baker oven dry, after pulverizing, crosses by comminutor 200 mesh sieves, accurately take by weighing be dried to constant weight sample 0.50 g in 50 ml centrifuge tubes, add the ultrasonic extraction of 700 W water-baths behind the 25 ml extracts, extract is pure water, extracting temperature is 30 ℃, extraction time is 40 minutes, extract once, after extract finishing centrifuge tube placed 20 ℃, centrifugal 10 min of hydro-extractor of 5000 r/min, draw supernatant 1 ml, behind 0.45 μ m water filtering with microporous membrane, directly inject sample injection bottle for analysis.
2.3. adenosine and cordycepin content are measured: precision is drawn adenosine and cordycepin mixed standard solution and fruiting bodies of cordyceps militaris test solution respectively, inject the sample introduction bottle after filtering, measure the fruiting bodies of cordyceps militaris test solution and should present the chromatographic peak identical with Cordycepin mixed standard solution chromatographic retention.
The mensuration of typical curve:
Get the adenosine of 6 variable concentrations that prepare and the cordycepin mixed standard solution vortex abundant mixing that vibrates, with directly injecting sample injection bottle behind the 0.45 μ m water filtering with microporous membrane.Each sample introduction repeats 3 times, and take concentration (μ g/mL) as horizontal ordinate, peak area is ordinate, makes the typical curve of measuring adenosine, cordycepin content, the results are shown in Figure 1.Figure 1A, Figure 1B are respectively the typical curve of adenosine and cordycepin in the embodiment of the invention.
The inventor has carried out the optimization of the inventive method analytical test condition by following manner.
(1) selection of extraction conditions
A. the investigation of extract concentration ratio
With pure water as solvent, choose in the extract that the ethanol volumetric concentration is respectively 0 %, 20 %, 40 %, 60 %, 80 %, 100 % test, acquired results is seen Fig. 2, as seen, the content of the adenosine and cordycepin in the fruiting bodies of cordyceps militaris surveyed obviously reduces along with the increase of concentration of alcohol in the extract among the figure.When concentration of alcohol was 100 %, adenosine and cordycepin content were extremely low.And when extract was pure water, both extraction content was the highest.Therefore, in the optimization method of the present invention, selection water is extract.
B, extraction time and the investigation of extracting temperature
Experiment finds that when extraction time was less than 30 min, institute's adenosine content of surveying had obvious raising with the increase of extraction time, and when extraction time was 40 min, the adenosine content of surveying reached maximum; Raise with temperature, the adenosine content of surveying has downtrending, and this may be owing to hydroxyl in the adenosine molecule is more, and under the higher condition of temperature, the interior or intermolecular easy formation hydrogen bond of adenosine molecule causes due to molecular structure changes.But in general, extraction time and temperature are all little on the impact of survey cordycepin.According to above experimental result, determine that in the more excellent scheme of the present invention, extraction times 40 min is Best Times, extract 30 ℃ of temperature and be the optimum extraction temperature.
C. extract the investigation of power and extraction time
Ultrasonic power in the leaching process changes less to the content influence of cordycepin, but with the raising of ultrasonic power, adenosine content slightly is improved.Therefore, this experimental selection experimental apparatus maximum ultrasonic power 700 W that can reach;
Extraction time is less on the impact of cordycepin and adenosine, this experimental result shows: water extracts 1 time, extracting 40 min can extract adenosine and cordycepin fully, extract and only make adenosine and cordycepin increase respectively by 0.044 % 2 times, 2.32 therefore % once extracts and can satisfy the analysis requirement, thereby, in the more excellent scheme of the inventive method, extraction time is for once.
(2), the investigation of mobile phase ratio in the chromatographic column
Adopt the methanol-water of different proportion as mobile phase, the separating effect of adenosine in the sample and cordycepin is compared, through repetition test, determine that finally first alcohol and water ratio is 16:84, flow velocity is 1.0 ml/min.
Present embodiment is also investigated precision and the stability of this method test, and is as follows:
(1), precision
Get adenosine and cordycepin mixed standard solution that mass concentration is 20 μ g/ml, excessively inject sample injection bottle and numbering behind the 0.45 μ m miillpore filter.Continuous sample introduction 6 times, record RSD value (the RSD value of adenosine and cordycepin, be relative standard deviation, be translated into relative standard deviation) be respectively 0.504 %, 0.442 %, all less than 2.0 %, when proof adopts this experimental technique that adenosine and cordycepin are carried out disposable quantitative test, favorable reproducibility, reliable results.The results are shown in following table 1:
Table 1
(2), the stability of adenosine and cordycepin content in the extract
Adopt extracting method after the above-mentioned optimization that one sample is carried out pre-treatment and obtain the fruiting bodies of cordyceps militaris need testing solution, at room temperature place respectively different time (0 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h), measure the content of its adenosine and cordycepin.Test is found: in 24 h, and the solution that adopts this experiment extraction conditions to extract, the content of its adenosine and cordycepin keeps relative stability, and the RSD value of six experiments of adenosine and cordycepin is respectively 4.11 % and 0.50 %.
(3), blank recovery experiment
Get adenosine and cordycepin mixed standard solution that mass concentration is 20 μ g/ml, excessively inject sample injection bottle behind the 0.45 μ m miillpore filter, continuous sample introduction 3 times, quantitative with typical curve.The blank recovery mean value that records adenosine and cordycepin is respectively 99.13%, 99.18%, illustrates that the chromatographic system detection sensitivity is high, good stability.
(4) recovery of standard addition experiment
Take by weighing fruiting bodies of cordyceps militaris dried powder 0.50 g, to 50 ml tool plug centrifuge tubes, add 15 ml ultrapure waters, jam-pack, ultrasonic extraction 40 min, be filtered in the 25 ml volumetric flasks, add 1.0 ml, 0.5 mg/ml adenosine and cordycepin mixed standard solution, constant volume, inject respectively sample injection bottle after crossing 0.45 μ m miillpore filter, numbering, the sample introduction analysis the results are shown in following table 2.The adenosine recovery is 94.30 % ~ 97.25 %, and average recovery rate is 95.78 %, and RSD is 1.54 %; The cordycepin recovery is 93.05 % ~ 95.35 %, and average recovery rate is 94.35 %, and RSD is 1.25 %.Illustrate that this experimental technique mark-on recovering effect is better, can be used for the assay of adenosine and cordycepin in the fruiting bodies of cordyceps militaris.
Table 2
Adopt the inventive method to test as follows to an artificial Cordyceps militaris sample of cultivating:
This artificial fruiting bodies of cordyceps militaris sample of cultivating is carried out pre-treatment, and concrete treatment step and liquid-phase condition are as previously described.Need testing solution 1 ml that absorption is handled well injects sample injection bottle and tests behind 0.45 μ m water filtering with microporous membrane, parallel processing 3 times, and the gained liquid chromatogram is as shown in Figure 3.Wherein the content of adenosine and cordycepin is respectively 162.81 mg/100 g, 148.03 mg/100 g, and the RSD value is respectively 2.125 %, 1.876 %.
Embodiment 2
The difference of present embodiment and embodiment 1 is that the extraction time of adenosine and cordycepin is 30min in the described fruiting bodies of cordyceps militaris.
The present invention has established the method that a kind of high performance liquid chromatography single injected sampling is measured adenosine and cordycepin in the fruiting bodies of cordyceps militaris simultaneously, and the sample extraction condition has been carried out more comprehensive optimization.The inventive method mainly contains following advantage:
1) with water as extract, simply pollution-free, the fruiting bodies of cordyceps militaris sample pre-treatments is simple, and is ultrasonic through water-bath, disposable extraction gets final product;
2) high, the good stability of the recovery.The recovery of standard addition of adenosine and cordycepin is respectively 95.78%, 94.35%, and the RSD of replication is respectively 3.311 %, 3.313 % (n=6);
3) analytical approach of more having reported has been simplified the composition of mobile phase, take the first alcohol and water as mobile phase, simply need not complicated process for preparation, has especially avoided the use of buffer salt to the damage of instrument, adopts isocratic elution, makes analytic process extremely easy.
The inventive method provides reference frame for the standard method of adenosine in the fruiting bodies of cordyceps militaris and cordycepin analysis.
Claims (10)
1. the method for adenosine and cordycepin content in the high-efficient liquid phase chromatogram technique analysis fruiting bodies of cordyceps militaris is characterized in that, comprises the steps:
(1) adenosine and cordycepin mixed standard solution preparation: take by weighing the adenosine and the cordycepin standard items that are dried to constant weight and place container, water dissolving and constant volume add adenosine and cordycepin mixed standard solution that ultrapure water is diluted to described standard reserving solution some variable concentrations as standard reserving solution;
Fruiting bodies of cordyceps militaris test solution preparation: fruiting bodies of cordyceps militaris is dried, through the comminutor grinding and sieving, accurately take by weighing the fruiting bodies of cordyceps militaris powdered sample that is dried to constant weight, add extract and extract, separate to get supernatant after extracting end, be the fruiting bodies of cordyceps militaris test solution;
(2) measure: draw respectively adenosine and cordycepin mixed standard solution and fruiting bodies of cordyceps militaris test solution, inject the sample introduction bottle after filtering, measure the fruiting bodies of cordyceps militaris test solution and should present the chromatographic peak identical with cordycepin mixed standard solution chromatographic retention with adenosine.
2. the method for adenosine and cordycepin content in the high-efficient liquid phase chromatogram technique analysis fruiting bodies of cordyceps militaris as claimed in claim 1, it is characterized in that, in the described step (1), the preparation method of described adenosine and cordycepin mixed standard solution is: accurately take by weighing adenosine and each 4mg of cordycepin standard items of being dried to constant weight, place 50 ml volumetric flasks, as standard reserving solution, concentration is designated as 80 μ g/ml with the water-soluble solution of 50ml and constant volume;
Described standard reserving solution is diluted to adenosine and the cordycepin mixed standard solution of 2 μ g/ml, 5 μ g/ml, 10 μ g/ml, 20 μ g/ml, 40 μ g/ml, 80 μ g/ml with ultrapure water.
3. the method for adenosine and cordycepin content in the high-efficient liquid phase chromatogram technique analysis fruiting bodies of cordyceps militaris as claimed in claim 2 is characterized in that, described accurately taking by weighing as being accurate to 0.0001 g.
4. the method for adenosine and cordycepin content in the high-efficient liquid phase chromatogram technique analysis fruiting bodies of cordyceps militaris as claimed in claim 1, it is characterized in that, in the described step (1), the fruiting bodies of cordyceps militaris test solution is prepared as: fruiting bodies of cordyceps militaris is placed 40 ℃ of drying baker oven dry, after pulverizing, crosses by comminutor 200 mesh sieves, accurately take by weighing be dried to constant weight sample 0.50 g in 50 ml centrifuge tubes, add the ultrasonic extraction of water-bath behind the 25 ml extracts, after extracting end centrifuge tube is placed 20 ℃, centrifugal 10 min in the hydro-extractor of 5000 r/min, draw supernatant 1 ml, behind 0.45 μ m water filtering with microporous membrane, directly inject sample injection bottle for analysis.
5. such as the method for adenosine and cordycepin content in claim 1 or the 4 described high-efficient liquid phase chromatogram technique analysis fruiting bodies of cordyceps militaris, it is characterized in that in the preparation of described step (1) fruiting bodies of cordyceps militaris test solution, described extract is pure water.
6. such as the method for adenosine and cordycepin content in claim 1 or the 4 described high-efficient liquid phase chromatogram technique analysis fruiting bodies of cordyceps militaris, it is characterized in that, in the preparation of described step (1) fruiting bodies of cordyceps militaris test solution, describedly be extracted as extraction once, extraction time is 30-40 minute, and extracting temperature is 30 ℃.
7. the method for adenosine and cordycepin content in the high-efficient liquid phase chromatogram technique analysis fruiting bodies of cordyceps militaris as claimed in claim 4 is characterized in that the ultrasonic power of the ultrasonic extraction of described water-bath is 700W.
8. the method for adenosine and cordycepin content in the high-efficient liquid phase chromatogram technique analysis fruiting bodies of cordyceps militaris as claimed in claim 1 is characterized in that in the described step (2), high-efficient liquid phase chromatogram condition is:
Chromatographic column is XBridgeTM C18 (5 μ m, 4.6 * 250 mm);
Mobile phase is methyl alcohol: water=16:84(v:v);
Flow velocity: 1.0 ml/min;
Sample size: 10 μ L, auto injection;
Column temperature: 30 ℃;
Detect wavelength: 254 nm.
9. the method for adenosine and cordycepin content in the high-efficient liquid phase chromatogram technique analysis fruiting bodies of cordyceps militaris as claimed in claim 8 is characterized in that the water in the described mobile phase is ultrapure water.
10. the method for adenosine and cordycepin content in the high-efficient liquid phase chromatogram technique analysis fruiting bodies of cordyceps militaris as claimed in claim 1 is characterized in that, described chromatographic determination process adopts isocratic elution.
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| CN103512975A (en) * | 2013-09-17 | 2014-01-15 | 上海交通大学 | Method for analyzing contents of effective substances in Cordyceps martialis fruiting body and residue by HPLC |
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| CN107817303A (en) * | 2017-10-17 | 2018-03-20 | 天津实发中科百奥工业生物技术有限公司 | The quick determination method of adenosine content in a kind of peacilomyce hepiahi bacterium silk powder |
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| CN113637042A (en) * | 2021-08-24 | 2021-11-12 | 杭州中非生物科技有限公司 | Preparation process and application of active components of silkworm cordyceps |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103512975A (en) * | 2013-09-17 | 2014-01-15 | 上海交通大学 | Method for analyzing contents of effective substances in Cordyceps martialis fruiting body and residue by HPLC |
| CN107817303A (en) * | 2017-10-17 | 2018-03-20 | 天津实发中科百奥工业生物技术有限公司 | The quick determination method of adenosine content in a kind of peacilomyce hepiahi bacterium silk powder |
| CN108125085A (en) * | 2017-11-24 | 2018-06-08 | 浙江工业大学 | A kind of cordyceps mycelia exocellular polysaccharide drinks and preparation method |
| CN107703233A (en) * | 2017-11-27 | 2018-02-16 | 威海百合生物技术股份有限公司 | A kind of detection method of adenosine content |
| CN110967413A (en) * | 2018-09-29 | 2020-04-07 | 泰州医药城国科化物生物医药科技有限公司 | Liquid phase separation analysis method for cordyceps militaris sporocarp and solid culture medium thereof |
| CN109406652A (en) * | 2018-11-01 | 2019-03-01 | 宜昌山城水都冬虫夏草有限公司 | A kind of detection method of Ecology cordyceps sinensis adenosine content |
| CN109521108A (en) * | 2018-11-08 | 2019-03-26 | 伊犁师范学院 | The detection method of cordycepin and adenosine in a kind of selenium-enriched cordceps militaris milk powder |
| CN110243989A (en) * | 2019-07-12 | 2019-09-17 | 山东奔月生物科技股份有限公司 | The liquid chromatography detecting method of cordycepin |
| CN113637042A (en) * | 2021-08-24 | 2021-11-12 | 杭州中非生物科技有限公司 | Preparation process and application of active components of silkworm cordyceps |
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