CN104694616A - Method for detecting content of adenosine and cordycepin in cordyceps militaris mycelium - Google Patents
Method for detecting content of adenosine and cordycepin in cordyceps militaris mycelium Download PDFInfo
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Abstract
The invention discloses a method for detecting the content of adenosine and cordycepin in a cordyceps militaris mycelium. The method particularly comprises the following steps: (1) repeatedly freezing and thawing the cordyceps militaris mycelium, then carrying out water bath ultrasonography, and taking filter liquor obtained after the water bath ultrasonography as a to-be-detected sample; and (2) respectively carrying out high-efficiency liquid chromatography detection on the obtained to-be-detected sample and a solution of the adenosine and/or a standard cordycepin product, and calculating to obtain the content of the adenosine and/or the cordycepin in the to-be-detected sample to according to a detection result. Compared with the traditional method, the method disclosed by the invention does not need to carry out drying treatment on a wet mycelium obtained through liquid fermentation and culture, can be used for simultaneously detecting the content of the adenosine and the cordycepin in the cordyceps militaris mycelium and detecting that the linear range of the adenosine is 2-50 micrograms/mL, the linear range of the cordycepin is 3-50 micrograms/mL, the detection limits of the adenosine and the cordycepin are respectively 1.1 micrograms/mL and 2.4 micrograms/mL and the recovery rate of two components is respectively 94%-100% and 99%-103%. The method disclosed by the invention has the advantages of time-saving property, energy conservation, high analysis speed and the like.
Description
Technical field
The present invention relates to a kind of method detecting the content of adenosine and cordycepin in cordyceps mycelium, particularly a kind of method of the content of adenosine and cordycepin in Cordyceps militaris (L.) Link. wet mycelium utilizing high performance liquid chromatography Simultaneously test supersound process to cross.
Background technology
Cordyceps militaris (L.) Link. (Cordyceps militaries) has another name called Cordyceps militaris, and widely distributed in China, Cordyceps militaris (L.) Link. has been selected into new resource for food catalogue as medicine-food two-purpose fungi.In recent years, people have been separated and have obtained various bioactivators from Cordyccps-militaris-(L.)-link. Sporophore and mycelia, mainly contain the compositions such as adenosine, cordycepin, Cordyceps polysaccharide, PEARLITOL 25C, SOD enzyme, sterols, have anti-inflammatory, anti-oxidant, antitumor, anticancer, improve the different physiological roles such as immunizing power.The content of the effective constituent such as adenosine, cordycepin in research display Cordyceps militaris (L.) Link. is suitable with wild cordyceps, has broad application prospects.
At present, Cordyceps militaris (L.) Link. artificial breeding technique is comparatively ripe, can be divided into solid state cultivation and liquid fermenting two kinds according to the difference of training method, and compared with solid state cultivation, liquid fermenting has cycle short, the advantage such as technique simply, is not easily polluted, mycelium production is large.As the important indicator of Cordyceps militaris (L.) Link. quality evalution, adenosine and cordycepin become the important indicative material of cordyceps mycelium fermentation culture, and therefore, detection method has great importance for instructing Cordyceps militaris Study on Fermentation fast and effectively.
At present, adenosine and cordycepin content utilize the method such as high performance liquid chromatography, capillary electrophoresis to detect, and the Cordyceps militaris (L.) Link. mensuration of adenosine and cordycepin content in mycelia that wets generally adopts and detects two kinds of component contents in dry mycelia and realize, there is length consuming time in wet mycelia drying process, big energy-consuming, composition be easy to the shortcomings such as change.As the people such as Niu Shuan measure (NIU Shuang to adenosine in cordyceps mycelium, 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.) adopt vacuum lyophilization as sample pre-treatments in, only determine a kind of component of adenosine, adenosine can not be detected simultaneously, cordycepin, and this method time consumption and energy consumption.
Summary of the invention
The object of this invention is to provide a kind of method detecting the content of adenosine and cordycepin in cordyceps mycelium.
In detection cordyceps mycelium provided by the present invention, the method for the content of adenosine and/or cordycepin, specifically can comprise the steps:
(1) water bath sonicator is carried out by after cordyceps mycelium multigelation, using ultrasonic rear filtrate as testing sample;
(2) by testing sample that step (1) obtains, and the solution of adenosine and/or cordycepin standard substance, carry out high performance liquid chromatography detection respectively under the same conditions, calculate the content of adenosine in described testing sample and/or cordycepin according to detected result.
In the step (1) of described method, the number of times of described multigelation can be 1-3 time (as 2-3 time); 1 described freeze thawing is: freezing 20-30min in liquid nitrogen, goes to the 50-60 DEG C of 10-15min that thaws.Further, for cordycepin, the number of times of described multigelation is better with 2-3 time.
In the step (1) of described method, the condition of described water bath sonicator is that 35-45KHz(is as 40KHz) continual ultrasonic, temperature can be 30-50 DEG C (as 40-50 DEG C), and the time can be 2-4h.Further, for adenosine, described temperature is better with 40-50 DEG C.
In the step (2) of described method, the condition of described high performance liquid chromatography is as follows: chromatographic column can be 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) (as 15:85) mixes; Type of elution is single moving phase wash-out, and elution time is 15min.
In the present invention, described chromatographic column is Klomasail C18(150mm × 4.6mm5 μm of particle size) chromatographic column.More concrete, be Sweden AKZO NOBEL Products, its catalog number is EH04713.
Further, in step (2), the flow velocity of described high performance liquid chromatography can be 1mL/min, and sample size can be 20 μ L, and column temperature can be 25 DEG C.
In the step (1) of described method, described cordyceps mycelium specifically obtains as follows: by Cordyceps militaris (L.) Link. fermented liquid 1600-1800g(as 1700g) centrifugal 15-20min, abandons supernatant, obtains described cordyceps mycelium.
Described Cordyceps militaris (L.) Link. fermented liquid is that the seed liquor of described cordyceps mycelium (cell concentration is 1g/100mL, and mycelia quality/bacteria liquid amasss) is inoculated in fermention medium, obtains after cultivating.The solvent of described fermention medium is water, solute and concentration as follows: sucrose 70g/L, soy peptone 30g/L, yeast extractive substance 30g/L, soybean cake powder 15g/L, K
2hPO
43H
2o2g/L, MgSO
47H
2o1g/L; PH is 7.Described cultivation is 25 DEG C of 200r/min shaking culture 7 days.The ratio of described inoculation is: the seed liquor of described cordyceps mycelium is 5% of the volume of described fermention medium.
The seed liquor of described cordyceps mycelium obtains as follows: by the strain inoculation of Cordyceps militaris (L.) Link. in seed culture medium, obtains after cultivating.The solvent of described seed culture medium is water, solute and concentration as follows: glucose 10g/L, yeast powder 10g/L, K
2hPO
43H
2o0.5g/L, KH
2pO
40.5g/L, MgSO
47H
2o0.5g/L; PH is 7.Described cultivation is 25 DEG C of 200r/min shaking culture 3 days.
In the step (1) of described method, after described cordyceps mycelium is carried out described multigelation, before described water bath sonicator, also comprise the step added water in described cordyceps mycelium, amount of water is the 40mL that adds water in the described cordyceps mycelium that obtains in Cordyceps militaris (L.) Link. fermented liquid described in every 8-10mL.
In the step (2) of described method, described ultrasonic rear filtrate obtains as follows: filter getting supernatant 0.45 μm of filtering membrane after ultrasonic rear centrifugal (20min as centrifugal in 1700g), gained filtrate is described ultrasonic rear filtrate.
In the step (2) of described method, the solution of described adenosine and/or cordycepin standard substance is the aqueous solution of adenosine and/or cordycepin standard substance.
Further, described adenosine and/or cordycepin standard substance specifically can be following any one:
(1) adenosine standard substance; (when described method is the method for the content detecting adenosine in cordyceps mycelium)
(2) cordycepin standard substance; (when described method is the method for the content detecting cordycepin in cordyceps mycelium)
(3) mixture of adenosine standard substance and cordycepin standard substance; (when described method is the method for the content simultaneously detecting adenosine and cordycepin in cordyceps mycelium)
More concrete, in the present invention, described adenosine standard substance are Sigma Products, and its catalog number is A925; Described cordycepin standard substance are Sigma Products, and its catalog number is C33941.
In the step (2) of described method, described " 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 under the same conditions; calculate the content of adenosine in described testing sample and/or cordycepin according to detected result ", specifically can be:
A1) the described adenosine of series concentration and/or the solution of cordycepin standard substance are carried out high performance liquid chromatography detection according to condition described above, with the concentration of described adenosine standard substance for X-coordinate, with the peak area of described adenosine standard substance for ordinate zou, draw adenosine typical curve; The testing sample that step (1) obtains is carried out high performance liquid chromatography detection according to condition described above, bring the peak area of the chromatographic peak with the identical retention time of described adenosine standard substance into described adenosine typical curve, obtain the content of the adenosine in described testing sample; And/or
A2) the described adenosine of series concentration and/or the solution of cordycepin standard substance are carried out high performance liquid chromatography detection according to condition described above, with the concentration of described cordycepin standard substance for X-coordinate, with the peak area of described cordycepin standard substance for ordinate zou, draw cordycepin typical curve; The testing sample that step (1) obtains is carried out high performance liquid chromatography detection according to condition described above, bring the peak area of the chromatographic peak with the identical retention time of described cordycepin standard substance into described cordycepin typical curve, obtain the content of the cordycepin in described testing sample.
At as above A1) and A2) in, when drawing described adenosine typical curve and described cordycepin typical curve, transverse and longitudinal coordinate can exchange.
At as above A1) and A2) in, the described adenosine of described series concentration and/or the solution of cordycepin standard substance are specially: solvent is water, the described adenosine standard substance of the quality such as solute is and described cordycepin standard substance, the series concentration of described adenosine standard substance and described cordycepin standard substance is 9 μ g/mL, 18 μ g/mL, 27 μ g/mL, 36 μ g/mL, 45 μ g/mL.
In the present invention, described Cordyceps militaris (L.) Link. is specially cordyceps militaris link bacterial strain CM-jd.
The method of the content of adenosine and cordycepin in detection cordyceps mycelium provided by the present invention, do not need compared with the conventional method to carry out drying treatment to the wet mycelia of liquid fermentation and culture, can adenosine and cordycepin content in Simultaneously test mycelium, the linearity range recording adenosine is 2-50 μ g/mL, the linearity range of cordycepin is 3-50 μ g/mL, the detection limit of adenosine and cordycepin is respectively 1.1 μ g/mL, 2.4 μ g/mL, and the rate of recovery of two kinds of compositions is respectively 94%-100%, 99%
-103%.The method has advantages such as saving time, energy-conservation, analysis speed is fast.
Accompanying drawing explanation
Fig. 1 is the HPLC color atlas of adenosine standard substance and cordycepin standard substance mixture.Wherein, peak 1 is adenosine standard substance (retention time is 5.314min), and peak 2 is cordycepin standard substance (retention time is 7.424min).
Fig. 2 is the HPLC color atlas of testing sample.Wherein, peak 1 is adenosine, and peak 2 is cordycepin.
Fig. 3 is the typical curve of adenosine.Wherein, the unit of X-coordinate concentration is μ g/mL.
Fig. 4 is the typical curve of cordycepin.Wherein, the unit of X-coordinate concentration is μ g/mL.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Instrument: Beijing Chuangxin Tongheng Science and Technology Co., Ltd. produces high performance liquid chromatograph, and model is P-3000, is equipped with UV-detector, and data processing and acquisition rely on CXTH-3000 workstation; Milli-Q ultrapure water system (Millipore Inc, Billerica, MA, USA); Electronic balance (BP210S, Sartorius, USA) very much; Supercentrifuge, ultrasonic apparatus;
Reagent: methyl alcohol (HPLC level, Fisher Scientific, USA); Adenosine standard substance (purity >=99%, is purchased from Sigma company, and its catalog number is A925); Cordycepin standard substance (purity >=99%, is purchased from Sigma company, and its catalog number is C33941);
Chromatographic column: Klomasail C18(150mm × 4.6mm, 5 μm of particle size), be Sweden AKZO NOBEL Products, its catalog number is EH04713.
Cordyceps militaris (L.) Link. (Cordyceps militaris) bacterial strain CM-jd: be recorded in " Wu Fengyao. the molecular structure that Cordyceps militaris (L.) Link. dwells on and biological activity research. Jiangsu University of Science and Technology, Master's thesis in 2011 " literary composition, the public can obtain from China Agricultural University.
The foundation of the content method of adenosine and cordycepin in embodiment 1, HPLC tracer liquid fermentation cordyceps mycelium
One, the fermentation culture of cordyceps mycelium
1, the acquisition of seed liquor
Seed culture medium: solvent is water, solute and concentration as follows: glucose 10g/L, yeast powder 10g/L, K
2hPO
43H
2o0.5g/L, KH
2pO
40.5g/L, MgSO
47H
2o0.5g/L; PH=7.
By the strain inoculation of Cordyceps militaris (L.) Link. (Cordyceps militaris) bacterial strain CM-jd in the 250mL shaking flask that 50mL seed culture medium is housed, 200r/min, cultivate 3 days for 25 DEG C, obtain seed liquor, measuring cell concentration is that 1g/100mL(mycelia quality/bacteria liquid amasss).
2, fermentation culture
Fermention medium: solvent is water, solute and concentration as follows: sucrose 70g/L, soy peptone 30g/L, yeast extractive substance 30g/L, soybean cake powder 15g/L, K
2hPO
43H
2o2g/L, MgSO
47H
2o1g/L; PH=7.
Step 1 is obtained seed liquor, according to 5%(volume fraction) inoculum size be inoculated into and be equipped with in the 250mL shaking flask of 50mL fermention medium, 200r/min, cultivate 7 days for 25 DEG C, obtain the fermented liquid of cordyceps mycelium.
Two, the optimization of ultrasonic extraction conditions
1, the optimization of cordyceps mycelium multigelation number of times
(1) testing sample is prepared
Be divided into 4 groups, often group get 8-10mL step one obtain Cordyceps militaris (L.) Link. fermented liquid, be placed in 50mL centrifuge tube, supercentrifuge be equivalent to 1700g with 3500r/min() the centrifugal 15-20min of rotating speed, abandoning supernatant, obtain cordyceps mycelium (precipitation).By mycelium freezing 20-30min in liquid nitrogen, be then placed in 50-60 DEG C of thermostat water bath and thaw 10-15min, (each group is respectively freeze thawing 0 time, 1 time, 2 times, 3 times to multigelation several times; Once freezing and once to thaw be a freeze thawing).40mL ultrapure water is added in centrifuge tube, after concussion evenly, 40 DEG C of water-bath 40KHz continual ultrasonic 3h.Liquid 3500r/min(after ultrasonic is equivalent to 1700g) after centrifugal 20min, get supernatant 0.45 μm of filtering membrane and filter, gained filtrate is testing sample, for high-performance liquid chromatogram determination.
(2) high-performance liquid chromatogram determination
The condition of high performance liquid chromatography is as follows: chromatographic column is Klomasail C18(150mm × 4.6mm, 5 μm of particle size); Column temperature is 25 DEG C, and sample size is 20 μ L; Moving phase is the solution that first alcohol and water mixes according to the ratio that volume ratio is 15:85; Type of elution is single moving phase wash-out, and elution time is 15min, and flow velocity is 1mL/min.
(3) drawing standard curve
Take adenosine standard substance and cordycepin standard substance respectively appropriate, dissolve with ultrapure water, be configured to the mother liquor that concentration is 100 μ g/mL respectively, then by appropriate two kinds of mother liquor mixing, adenosine standard substance are diluted to and cordycepin standard substance final concentration is 9 μ g/mL with ultrapure water, 18 μ g/mL, 27 μ g/mL, 36 μ g/mL, the standard working solution of 45 μ g/mL, the standard working solution of the HPLC condition of step (2) to as above series concentration is utilized to measure, with two kinds of standard substance peak area separately, regression analysis is done to concentration respectively, namely with the concentration (μ g/mL) of adenosine standard substance (or cordycepin standard substance) standard working solution for X-coordinate, with the peak area of adenosine standard substance (or cordycepin standard substance) for ordinate zou, drawing standard curve.Finally obtain adenosine typical curve and cordycepin typical curve.
(4) analysis of data
The HPLC collection of illustrative plates of testing sample and the color atlas of adenosine standard substance and cordycepin standard substance are contrasted, the chromatographic peak peak area consistent with adenosine standard substance retention time is brought into the adenosine typical curve that step (3) obtains, thus calculate the content of adenosine in testing sample; The chromatographic peak peak area consistent with cordycepin standard substance retention time is brought into the cordycepin typical curve that step (3) obtains, thus calculate the content of cordycepin in testing sample.
Test in triplicate, results averaged.
Result shows, and in HPLC color atlas, the retention time of adenosine standard substance is 5.314min, and the retention time of cordycepin standard substance is 7.424min(Fig. 1).Different number of times multigelation, in the testing sample finally recorded, the content of adenosine and cordycepin is as shown in table 1.Visible, no matter be adenosine or cordycepin, freeze thawing group is all than non-freeze thawing group ultrasonic extracting method good (P<0.05).For adenosine, number of freezing and thawing is that the ultrasonic extracting method difference of 1-3 time is not remarkable; For cordycepin, number of freezing and thawing is that to be better than number of freezing and thawing be 1 time (P<0.05) to the ultrasonic extracting method of 2-3 time.
The content of adenosine and cordycepin in the testing sample that the different number of times multigelation of table 1 records
Note: significant difference in the level that in table, in colleague, different lowercase alphabet is shown in P<0.05.
2, the optimization of cordyceps mycelium ultrasonic temperature
(1) testing sample is prepared
Be divided into 4 groups, often group get 8-10mL step one obtain Cordyceps militaris (L.) Link. fermented liquid, be placed in 50mL centrifuge tube, supercentrifuge be equivalent to 1700g with 3500r/min() the centrifugal 15-20min of rotating speed, abandoning supernatant, obtain cordyceps mycelium (precipitation).By mycelium freezing 20-30min in liquid nitrogen, be then placed in 50-60 DEG C of thermostat water bath and thaw 10-15min, freeze thawing 2 times altogether (once freezing and once thaw be a freeze thawing).In centrifuge tube, add 40mL ultrapure water, after concussion evenly, each group respectively at 20 DEG C, 30 DEG C, 40 DEG C, 50 DEG C water-bath 40KHz continual ultrasonic 3h.Liquid 3500r/min(after ultrasonic is equivalent to 1700g) after centrifugal 20min, get supernatant 0.45 μm of filtering membrane and filter, gained filtrate is testing sample, for high-performance liquid chromatogram determination.
(2) high-performance liquid chromatogram determination
With step 2 1(2).
(3) drawing standard curve
With step 2 1(3).
(4) analysis of data
With step 2 1(4).
Test in triplicate, results averaged.
Result shows, and in HPLC color atlas, the retention time of adenosine standard substance is 5.314min, and the retention time of cordycepin standard substance is 7.424min(Fig. 1).Different ultrasonic temperature, in the testing sample finally recorded, the content of adenosine and cordycepin is as shown in table 2.Visible, no matter be adenosine or cordycepin, when ultrasonic temperature is 30-50 DEG C, ultrasonic extracting method is all better than 20 DEG C of groups (P<0.05).For adenosine, ultrasonic temperature is that the ultrasonic extracting method of 40-50 DEG C is better than 30 DEG C (P<0.05); For cordycepin, ultrasonic temperature is that the ultrasonic extracting method difference of 30-50 DEG C is not remarkable.
The content of adenosine and cordycepin in the testing sample recorded under the different ultrasonic temperature of table 2
Note: significant difference in the level that in table, in colleague, different lowercase alphabet is shown in P<0.05.
3, the optimization of cordyceps mycelium ultrasonic time
(1) testing sample is prepared
Be divided into 4 groups, often group get 8-10mL step one obtain Cordyceps militaris (L.) Link. fermented liquid, be placed in 50mL centrifuge tube, supercentrifuge be equivalent to 1700g with 3500r/min() the centrifugal 15-20min of rotating speed, abandoning supernatant, obtain cordyceps mycelium (precipitation).By mycelium freezing 20-30min in liquid nitrogen, be then placed in 50-60 DEG C of thermostat water bath and thaw 10-15min, freeze thawing 2 times altogether (once freezing and once thaw be a freeze thawing).In centrifuge tube, add 40mL ultrapure water, after concussion evenly, 40 DEG C of water-bath 40KHz continual ultrasonic, the ultrasonic time of each group is respectively 1h, 2h, 3h, 4h.Liquid 3500r/min(after ultrasonic is equivalent to 1700g) after centrifugal 20min, get supernatant 0.45 μm of filtering membrane and filter, gained filtrate is testing sample, for high-performance liquid chromatogram determination.
(2) high-performance liquid chromatogram determination
With step 2 1(2).
(3) drawing standard curve
With step 2 1(3).
(4) analysis of data
With step 2 1(4).
Test in triplicate, results averaged.
Result shows, and in HPLC color atlas, the retention time of adenosine standard substance is 5.314min, and the retention time of cordycepin standard substance is 7.424min(Fig. 1).Different ultrasonic time, in the testing sample finally recorded, the content of adenosine and cordycepin is as shown in table 3.Visible, when ultrasonic time is 2-4h, ultrasonic extracting method is better than other each group (P<0.05).
The content of adenosine and cordycepin in the testing sample recorded under the different ultrasonic time of table 3
Note: significant difference in the level that in table, in colleague, different lowercase alphabet is shown in P<0.05.
The optimum result of comprehensive above each parameter, multigelation number of times during visible supersound process is advisable with 1-3 time, and 2-3 time better, and ultrasonic temperature is advisable with 30-50 DEG C, and 40-50 DEG C better, and ultrasonic time is advisable with 2-4h.Take into account the principle of saving while considering test effect, multigelation number of times when determining supersound process in follow-up test adopts 2 times, and ultrasonic temperature adopts 40 DEG C, and ultrasonic time adopts 2h.
Three, the rate of recovery of adenosine and cordycepin is measured under condition after optimization
(1) testing sample is prepared
Get the Cordyceps militaris (L.) Link. fermented liquid that two parts of 5mL steps one obtain, be placed in 50mL centrifuge tube (being numbered 1 and 2 respectively) respectively, supercentrifuge is equivalent to 1700g with 3500r/min() the centrifugal 15-20min of rotating speed, abandoning supernatant, obtain cordyceps mycelium (precipitation).By two parts of mycelium freezing 20-30min in liquid nitrogen, be then placed in 50-60 DEG C of thermostat water bath and thaw 10-15min, freeze thawing 2 times altogether (once freezing and once thaw be a freeze thawing).In the centrifuge tube of No. 1 sample, add 0.08mL adenosine mother liquor (concentration is 50 μ g/mL), 0.2mL cordycepin mother liquor (concentration is 100 μ g/mL) respectively, then add 40mL ultrapure water; And in the centrifuge tube of No. 2 samples, additionally do not add adenosine and cordycepin, only add 40mL ultrapure water.After No. 1 sample hose and No. 2 sample hose concussions evenly, all in 40 DEG C of water-bath 40KHz continual ultrasonic 2h.Liquid 3500r/min(after ultrasonic is equivalent to 1700g) after centrifugal 20min, get supernatant 0.45 μm of filtering membrane and filter, gained filtrate is testing sample, for high-performance liquid chromatogram determination.Wherein, No. 1 sample hose gained filtrate is considered as adding rear sample; No. 2 sample hose gained filtrates are considered as adding front sample.
(2) high-performance liquid chromatogram determination
With step 2 1(2).
(3) drawing standard curve
With step 2 1(3).
(4) analysis of data
With reference to step 2 1(4), calculate the content of sample and the adenosine after adding in sample and cordycepin before adding respectively.And then, according to the rate of recovery of following formulae discovery adenosine and cordycepin, content/(before adding in sample adenosine or the content+interpolation adenosine of cordycepin or the amount of cordycepin) × 100% of adenosine or cordycepin in sample after the rate of recovery=interpolation.
Experiment in triplicate.
Result is as shown in table 4, and the rate of recovery of adenosine and cordycepin two kinds of compositions can reach respectively for 94%-100%, 99%-103%.In the visible HPLC tracer liquid fermentation cordyceps mycelium as above provided, the content method result of adenosine and cordycepin accurately and reliably.
The mensuration (unit: %) of the rate of recovery of table 4 adenosine and cordycepin
The content of adenosine and cordycepin in condition tracer liquid fermentation cordyceps mycelium after embodiment 2, the optimization of employing embodiment 1
One, the fermentation culture of cordyceps mycelium
With embodiment 1 step one.
Two, the supersound extraction under postcondition and HPLC detection is optimized
(1) testing sample is prepared
Get 8-10mL step one obtain Cordyceps militaris (L.) Link. fermented liquid, be placed in 50mL centrifuge tube, supercentrifuge be equivalent to 1700g with 3500r/min() the centrifugal 15-20min of rotating speed, abandoning supernatant, obtain cordyceps mycelium (precipitation).By mycelium freezing 20-30min in liquid nitrogen, be then placed in 50-60 DEG C of thermostat water bath and thaw 10-15min, freeze thawing 2 times altogether (once freezing and once thaw be a freeze thawing).40mL ultrapure water is added in centrifuge tube, after concussion evenly, 40 DEG C of water-bath 40KHz continual ultrasonic 2h.Liquid 3500r/min(after ultrasonic is equivalent to 1700g) after centrifugal 20min, get supernatant 0.45 μm of filtering membrane and filter, gained filtrate is testing sample, for high-performance liquid chromatogram determination.
(2) high-performance liquid chromatogram determination
With embodiment 1 step 2 1(2).
(3) drawing standard curve
With embodiment 1 step 2 1(3).
(4) analysis of data
With embodiment 1 step 2 1(4).
Test in triplicate, results averaged.
Result shows:
1. in HPLC color atlas, the retention time of adenosine standard substance is 5.314min, and the retention time of cordycepin standard substance is 7.424min(Fig. 1).Obvious chromatographic peak (Fig. 2) is all there is in testing sample at the retention time place identical with cordycepin standard substance with adenosine standard substance.
2. as shown in Figure 3 and Figure 4, the linearity range recording adenosine is 2-50 μ g/mL, and the linearity range of cordycepin is 3-50 μ g/mL, and gained peak area-concentration standard curve is respectively:
Adenosine: Y=-1.191 × 10
5+ 1.042 × 10
5x, r=0.998931;
Cordycepin: Y=-3.567 × 10
5+ 1.470 × 10
5x, r=0.997197;
3. the detection limit of adenosine and cordycepin is respectively 1.1 μ g/mL, 2.4 μ g/mL(when in typical curve during Y=0, the value of X).
The adenosine of final mensuration and the sample concentration mean value of cordycepin are respectively 1.65mg/L and 4.30mg/L(table 5).The content of adenosine and cordycepin in condition tracer liquid fermentation cordyceps mycelium after table 5 adopts embodiment 1 to optimize
| Repeat 1 | Repeat 2 | Repeat 3 | Mean value | |
| 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.
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