CN104090038A - Method for directly measuring content of cordyceps sinensis polysaccharide peptide in cordyceps sinensis product - Google Patents
Method for directly measuring content of cordyceps sinensis polysaccharide peptide in cordyceps sinensis product Download PDFInfo
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- CN104090038A CN104090038A CN201410320033.4A CN201410320033A CN104090038A CN 104090038 A CN104090038 A CN 104090038A CN 201410320033 A CN201410320033 A CN 201410320033A CN 104090038 A CN104090038 A CN 104090038A
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- cordyceps sinensis
- caterpillar fungus
- chinese caterpillar
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Abstract
The invention relates to a method for simply, quickly, directly and accurately measuring the connect of cordyceps sinensis polysaccharide peptide in a cordyceps sinensis product. The method comprises the following steps: performing simple pretreatment on a sample, and qualitatively and quantitatively analyzing through a high performance liquid chromatography photo-diode array (HPLC-PDA) detector. By utilizing the advantages of accurate quantification of high performance liquid chromatography, accurate spectral analysis qualification of the PDA detector and the like, the method can be used for simply, quickly, accurately and directly measuring the connect of cordyceps sinensis polysaccharide peptide in the ordyceps sinensis product, and a simple, reliable and easily-popularized method is supplied to authentication analysis, quality evaluation and quality control of health efficacy factors in the cordyceps sinensis product.
Description
Technical field
The invention belongs to Food Inspection and analysis field, be specially a kind of method that the HPLC-PDA of employing method is measured Chinese caterpillar fungus polysaccharide peptide content in Cordyceps sinensis product accurately and rapidly.
Background technology
Cordyceps sinensis (formal name used at school: Ophiocordyceps sinensis), have another name called cordyceps sinensis, be rare traditional Chinese medicine traditional in Chinese history, mainly originate in Qinghai, China's Mainland, Tibet, Sichuan, Yunnan, Gansu and Guizhou etc. and economize and municipal severe cold areas and snow-capped mountains and marshlands.Modern medicine proof Cordyceps sinensis has calmness, relieving cough and reducing sputum and inhibition tumor cell is grown, increase body has the effect of peculiar immunity to fitness and disease prevention, and Chinese caterpillar fungus polysaccharide peptide is exactly one of main composition wherein.Chinese caterpillar fungus polysaccharide peptide is a kind of glycoprotein, is the combination of polysaccharide and protein, has very high molecular weight, the repetitive of disaccharide, consists of.Chinese caterpillar fungus polysaccharide peptide has very strong biological activity, and Primary Study polysaccharide Toplink enhanced machine body immunity function, has antitumor action and antioxidation.But, yet there are no, the easily record of universal detection method quick, accurate to Chinese caterpillar fungus polysaccharide peptide in Cordyceps sinensis product.
Summary of the invention
For addressing the above problem, the invention provides the method for Chinese caterpillar fungus polysaccharide peptide content in a kind of direct mensuration Cordyceps sinensis product, its technical scheme adopting is as follows:
A method for Chinese caterpillar fungus polysaccharide peptide content in direct mensuration Cordyceps sinensis product, is characterized in that: said method comprising the steps of:
(1) sample preparation
Take Cordyceps sinensis product appropriate, after adding water, carry out water-bath extraction, after it is cooling, adding water constant volume, to become concentration be the Cordyceps sinensis product water solution of 5.00mg/ml, filter this solution, get part filtrate and add after absolute ethyl alcohol centrifugally by hydro-extractor, then taking precipitate adopts mobile phase to dissolve, and crosses after organic filter membrane to such an extent that sample solution is standby;
(2) mensuration of high performance liquid chromatograph
Step (one) gained sample solution is got in right amount and joined in high performance liquid chromatograph and measure, and measurement result is carried out to qualitative analysis;
(3) formulation of typical curve
Precision take be dried to constant weight Chinese caterpillar fungus polysaccharide poly saccharide peptide standard product 0.0500g in 10ml volumetric flask, water dissolves and constant volume, the concentration Chinese caterpillar fungus polysaccharide peptide standard reserve liquid that is 5.00mg/ml, then get respectively 100 μ l, 200 μ l, 300 μ l, 400 μ l, 500 μ l standard reserve liquids are in 5ml volumetric flask, add water constant volume, obtain concentration and be respectively 100.0 μ g/ml, 200.0 μ g/ml, 300.0 μ g/ml, 400.0 μ g/ml, the standard operation liquid of 500.0 μ g/ml, standard operation liquid is measured by high performance liquid chromatograph, the data obtained be take concentration as horizontal ordinate, peak area is ordinate, set up concentration-peak area typical curve, and draw standard equation.
(4) the determining of Chinese caterpillar fungus polysaccharide peptide content in sample
The linear equation drawing according to concentration-peak area typical curve, is scaled concentration by the peak area of Chinese caterpillar fungus polysaccharide peptide measured in sample solution in step (two), thereby calculates the Chinese caterpillar fungus polysaccharide peptide content in this Cordyceps sinensis product.
Further, when high performance liquid chromatograph is measured, liquid phase chromatogram condition is: chromatographic column is Atlantis T3 chromatographic column, its specification is 4.6 * 250mm, i.d..5 μ m, and column temperature is 35 ℃, and mobile phase is that volume fraction is 5% methanol aqueous solution, flow velocity is 1ml/min, sample size is 10 μ l, and detecting device is diode array detector, and scanning wavelength is 190nm-460nm.
Further, the temperature that described water-bath is extracted is 60 ℃, and the time is 30 minutes.
Further, the rotating speed of hydro-extractor used is 9000rpm, and centrifugation time is 15 minutes.
Further, organic filter membrane used is the organic filter membrane of 0.22 μ m.
Further, described Cordyceps sinensis product comprises Cordyceps sinensis medicinal raw material and Chinese caterpillar fungus health-care food.
By the realization of technique scheme, the invention provides a kind of accurate, easy, quick, easy method of Chinese caterpillar fungus polysaccharide peptide in universal mensuration Cordyceps sinensis product, the present invention carries out qualitative and quantitative analysis by high performance liquid chromatography diode array detector to sample, pre-treatment is simple and convenient, without the separated impurity removing technology that waits of loaded down with trivial details extraction, for discriminatory analysis, quality evaluation and the quality control of Chinese caterpillar fungus polysaccharide peptide efficacy factor in Cordyceps sinensis product provides effective way and reliable method.
Accompanying drawing explanation
Fig. 1 is the chromatogram of Chinese caterpillar fungus polysaccharide poly saccharide peptide standard product in the present invention;
Fig. 2 is the spectrogram of Chinese caterpillar fungus polysaccharide poly saccharide peptide standard product in the present invention;
Fig. 3 is the chromatogram of Chinese caterpillar fungus polysaccharide peptide in sample;
Fig. 4 is the spectrogram of Chinese caterpillar fungus polysaccharide peptide in sample;
Fig. 5 is concentration-peak area typical curve of Chinese caterpillar fungus polysaccharide peptide.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in more detail.
Embodiment 1
Adopt method provided by the present invention to measure the good Chinese caterpillar fungus polysaccharide peptide of converting in the pure powder sample of Cordyceps sinensis in Qinghai.
1, measuring the instrument and the medicine that adopt has: Waterse2695 type high performance liquid chromatograph system, Waterse2996 type diode array detector, Empower chromatographic work station and data handling system; Chinese caterpillar fungus polysaccharide poly saccharide peptide standard product (> 98%, state's house fungus grass Engineering Technical Research Centre);
2, determination step is as follows:
(1) sample preparation
Take the good pure powder sample of the Cordyceps sinensis 0.25g that converts in Qinghai, after adding water 10ml, in 60 ℃ of water-baths, extract 30min, after it is cooling, add water and be settled to 50ml, becoming concentration is the worm summer grass product aqueous solution of 5.00mg/ml, filter this solution, get 2ml filtrate, add after 6ml absolute ethyl alcohol by the centrifugal 15min of hydro-extractor, the rotating speed of hydro-extractor is 9000rpm, then taking precipitate adopts the methanol aqueous solution that volume fraction is 5% to dissolve, and excessively after the organic filter membrane of 0.22 μ m, obtains sample solution standby;
(2) mensuration of high performance liquid chromatograph
Step (one) gained sample solution is got in right amount and joined in high performance liquid chromatograph and measure, the liquid phase chromatogram condition adopting is: chromatographic column is Atlantis T3 chromatographic column, its specification is 4.6 * 250mm, i.d..5 μ m, column temperature is 35 ℃, mobile phase is that volume fraction is 5% methanol aqueous solution, and flow velocity is 1ml/min, and sample size is 10 μ l, detecting device is diode array detector, and scanning wavelength is 190nm-460nm.The principle of work of diode array tube detecting device is to enter injection device after polychromatic light is absorbed by compositional selecting by sample cell again, be radiated at and on diode array device, make the light intensity of each nano wave length be transformed into corresponding electrical signal intensity, obtain the absorption spectrum of component.Therefore, can be tentatively more qualitative to compound with standard substance retention time according to the chromatographic retention of object (i.e. time when sample introduction starts to occur this concentration of component maximum value to post), utilize the absorption spectrum of object and standard substance more qualitative simultaneously.As shown in Figure 3, in sample solution, the retention time of certain component is 2.534 minutes, in this and Fig. 1, the retention time 2.601 of Chinese caterpillar fungus polysaccharide poly saccharide peptide standard product approaches, and belongs to normal error range, therefore can determine that this component in sample solution is Chinese caterpillar fungus polysaccharide peptide; As shown in Figure 4, within the scope of scanning wavelength 190nm-460nm, in sample solution, the maximum absorption wavelength of certain component is 192.3nm, and this is identical with the maximum absorption wavelength 192.3nm of Chinese caterpillar fungus polysaccharide poly saccharide peptide standard product in Fig. 2, can further determine that this component is Chinese caterpillar fungus polysaccharide peptide; Like this dual qualitative more accurately and reliably, can more directly, more promptly detect in sample, whether to contain peptidoglycan.
(3) formulation of typical curve
Precision take be dried to constant weight Chinese caterpillar fungus polysaccharide poly saccharide peptide standard product 0.0500g in 10ml volumetric flask, water dissolves and constant volume, the concentration Chinese caterpillar fungus polysaccharide peptide standard reserve liquid that is 5.00mg/ml, then get respectively 100 μ l, 200 μ l, 300 μ l, 400 μ l, 500 μ l standard reserve liquids are in 5ml volumetric flask, add water constant volume, obtain concentration and be respectively 100.0 μ g/ml, 200.0 μ g/ml, 300.0 μ g/ml, 400.0 μ g/ml, the standard operation liquid of 500.0 μ g/ml, standard operation liquid is measured by high performance liquid chromatograph, as Fig. 5, the data obtained be take concentration as horizontal ordinate, peak area is ordinate, set up concentration-peak area typical curve, and draw standard equation: Y=1.17e
0.05x+2.56e
0.04, degree of fitting R
2=0.99996, the recovery is 96.3%, detects and is limited to 0.1mg/kg.
(4) the determining of Chinese caterpillar fungus polysaccharide peptide content in sample
Standard equation by the peak area of measured Chinese caterpillar fungus polysaccharide peptide in sample solution in step (two) for people's step (three) gained, being converted into the concentration of Chinese caterpillar fungus polysaccharide peptide, is 16.8mg/g thereby calculate the good content of converting Chinese caterpillar fungus polysaccharide peptide in the pure powder sample of Cordyceps sinensis in Qinghai.
These are only better embodiment of the present invention, all equalizations of doing according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (6)
1. directly measure a method for Chinese caterpillar fungus polysaccharide peptide content in Cordyceps sinensis product, it is characterized in that: said method comprising the steps of:
(1) sample preparation
Take Cordyceps sinensis product appropriate, after adding water, carry out water-bath extraction, after it is cooling, adding water constant volume, to become concentration be the worm summer grass product aqueous solution of 5.00mg/ml, filter this solution, get part filtrate and add after absolute ethyl alcohol centrifugally by hydro-extractor, then taking precipitate adopts mobile phase to dissolve, and crosses after organic filter membrane to such an extent that sample solution is standby;
(2) mensuration of high performance liquid chromatograph
Step (one) gained sample solution is got in right amount and joined in high performance liquid chromatograph and measure, and measurement result is carried out to qualitative analysis;
(3) formulation of typical curve
Precision take be dried to constant weight Chinese caterpillar fungus polysaccharide poly saccharide peptide standard product 0.0500g in 10ml volumetric flask, water dissolves and constant volume, the concentration Chinese caterpillar fungus polysaccharide peptide standard reserve liquid that is 5.00mg/ml, then get respectively 100 μ l, 200 μ l, 300 μ l, 400 μ l, 500 μ l standard reserve liquids are in 5ml volumetric flask, add water constant volume, obtain concentration and be respectively 100.0 μ g/ml, 200.0 μ g/ml, 300.0 μ g/ml, 400.0 μ g/ml, the standard operation liquid of 500.0 μ g/ml, standard operation liquid is measured by high performance liquid chromatograph, the data obtained be take concentration as horizontal ordinate, peak area is ordinate, set up concentration-peak area typical curve, and draw standard equation.
(4) the determining of Chinese caterpillar fungus polysaccharide peptide content in sample
The linear equation drawing according to concentration-peak area typical curve, is scaled concentration by the peak area of Chinese caterpillar fungus polysaccharide peptide measured in sample solution in step (two), thereby calculates the Chinese caterpillar fungus polysaccharide peptide content in this Cordyceps sinensis product.
2. a kind of method of Chinese caterpillar fungus polysaccharide peptide content in direct mensuration Cordyceps sinensis product as claimed in claim 1, it is characterized in that: when high performance liquid chromatograph is measured, liquid phase chromatogram condition is: chromatographic column is Atlantis T3 chromatographic column, its specification is 4.6 * 250mm, i.d..5 μ m, and column temperature is 35 ℃, and mobile phase is that volume fraction is 5% methanol aqueous solution, flow velocity is 1ml/min, sample size is 10 μ l, and detecting device is diode array detector, and scanning wavelength is 190nm-460nm.
3. a kind of method of Chinese caterpillar fungus polysaccharide peptide content in direct mensuration Cordyceps sinensis product as claimed in claim 1, is characterized in that: the temperature that described water-bath is extracted is 60 ℃, and the time is 30 minutes.
4. a kind of method of Chinese caterpillar fungus polysaccharide peptide content in direct mensuration Cordyceps sinensis product as claimed in claim 1, is characterized in that: the rotating speed of hydro-extractor used is 9000rpm, and centrifugation time is 15 minutes.
5. a kind of method of Chinese caterpillar fungus polysaccharide peptide content in direct mensuration Cordyceps sinensis product as claimed in claim 1, is characterized in that: organic filter membrane used is the organic filter membrane of 0.22 μ m.
6. a kind of method of Chinese caterpillar fungus polysaccharide peptide content in direct mensuration Cordyceps sinensis product as claimed in claim 1, is characterized in that: described Cordyceps sinensis product comprises Cordyceps sinensis medicinal raw material and Chinese caterpillar fungus health-care food.
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| CN107110831A (en) * | 2014-11-18 | 2017-08-29 | 弗特克斯药品有限公司 | The method for carrying out high throughput test high performance liquid chromatography |
| US10626111B2 (en) | 2004-01-30 | 2020-04-21 | Vertex Pharmaceuticals Incorporated | Modulators of ATP-binding cassette transporters |
| US11052075B2 (en) | 2010-04-07 | 2021-07-06 | Vertex Pharmaceuticals Incorporated | Pharmaceutical compositions of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl) cyclopropanecarboxamido)-3-methylpyridin-2-yl) benzoic acid and administration thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10626111B2 (en) | 2004-01-30 | 2020-04-21 | Vertex Pharmaceuticals Incorporated | Modulators of ATP-binding cassette transporters |
| US11084804B2 (en) | 2005-11-08 | 2021-08-10 | Vertex Pharmaceuticals Incorporated | Modulators of ATP-binding cassette transporters |
| US11052075B2 (en) | 2010-04-07 | 2021-07-06 | Vertex Pharmaceuticals Incorporated | Pharmaceutical compositions of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl) cyclopropanecarboxamido)-3-methylpyridin-2-yl) benzoic acid and administration thereof |
| CN107110831A (en) * | 2014-11-18 | 2017-08-29 | 弗特克斯药品有限公司 | The method for carrying out high throughput test high performance liquid chromatography |
| CN104569406A (en) * | 2014-12-19 | 2015-04-29 | 深圳市计量质量检测研究院 | Kit for enzyme-linked immunosorbent assay of cordyceps sinensis and preparation method of kit |
| CN104569406B (en) * | 2014-12-19 | 2016-09-14 | 深圳市计量质量检测研究院 | A kind of Cordyceps enzyme-linked immunologic detecting kit and preparation method thereof |
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