CN104090038B - A kind of method of Chinese caterpillar fungus polysaccharide peptide content in direct mensuration Cordyceps sinensis product - Google Patents
A kind of method of Chinese caterpillar fungus polysaccharide peptide content in direct mensuration Cordyceps sinensis product Download PDFInfo
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- CN104090038B CN104090038B CN201410320033.4A CN201410320033A CN104090038B CN 104090038 B CN104090038 B CN 104090038B CN 201410320033 A CN201410320033 A CN 201410320033A CN 104090038 B CN104090038 B CN 104090038B
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- caterpillar fungus
- chinese caterpillar
- cordyceps sinensis
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- polysaccharide peptide
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Abstract
The present invention relates to the method for Chinese caterpillar fungus polysaccharide peptide content in a kind of easy, quick, direct Accurate Determining Cordyceps sinensis product.Sample utilizes high performance liquid chromatography diode array detector (HPLC-PDA) to carry out quantification and qualification after simple pre-treatment.The method uses high performance liquid chromatography quantitatively accurately and the qualitative advantage such as accurately of PDA detecting device spectral analysis, simple and quick, directly to measure Chinese caterpillar fungus polysaccharide peptide in Cordyceps sinensis product exactly content, for the discriminatory analysis of the health-care efficacy factor in Cordyceps sinensis product, quality evaluation and quality control provide easy, reliable, easily universal method.
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 measures 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 proves that Cordyceps sinensis has calmness, the growth of relieving cough and reducing sputum and inhibition tumor cell, increases body has peculiar immunity effect 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, is made up of the repetitive of disaccharide.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 record of the detection method that Chinese caterpillar fungus polysaccharide peptide in Cordyceps sinensis product fast, accurately, is easily popularized.
Summary of the invention
For solving the 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 adopted 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, water-bath extraction is carried out after adding water, after its cooling, the constant volume that adds water becomes concentration to be the Cordyceps sinensis product water solution of 5.00mg/ml, filter this solution, getting portion filtrate adds by centrifuge after absolute ethyl alcohol, and then taking precipitate adopts mobile phase to dissolve, and to cross after organic filter membrane sample solution is for subsequent use;
(2) mensuration of high performance liquid chromatograph
Step (one) gained sample solution is got to join in high performance liquid chromatograph in right amount and measures, and qualitative analysis is carried out to measurement result;
(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, by water-soluble solution and constant volume, obtain the Chinese caterpillar fungus polysaccharide peptide standard reserve liquid that concentration is 5.00mg/ml, then 100 μ l are got respectively, 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 working solution of 500.0 μ g/ml, standard working solution is measured by high performance liquid chromatograph, the data obtained take concentration as horizontal ordinate, peak area is ordinate, set up concentration-peak area typical curve, and draw standard equation.
(4) determination of Chinese caterpillar fungus polysaccharide peptide content in sample
According to the linear equation that concentration-peak area typical curve draws, the peak area of Chinese caterpillar fungus polysaccharide peptide measured in sample solution in step (two) is scaled concentration, thus calculates the Chinese caterpillar fungus polysaccharide peptide content in this Cordyceps sinensis product.
Further, when high performance liquid chromatograph measures, liquid phase chromatogram condition is: chromatographic column is Atlantis T3 chromatographic column, its specification is 4.6 × 250mm, i.d..5 μm, and column temperature is 35 DEG C, mobile phase to be volume fraction be 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 DEG C, 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 0.22 μm of organic filter membrane.
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 the method for Chinese caterpillar fungus polysaccharide peptide in a kind of accurate, easy, quick, easily universal mensuration Cordyceps sinensis product, the present invention carries out quantification and qualification by high performance liquid chromatography diode array detector to sample, pre-treatment is simple and convenient, without the need to impurity removing technologies such as loaded down with trivial details extraction and isolation, for the discriminatory analysis of Chinese caterpillar fungus polysaccharide peptide efficacy factor in Cordyceps sinensis product, quality evaluation and quality control provide 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 the 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
Method provided by the present invention is adopted to measure the good Chinese caterpillar fungus polysaccharide peptide converted in the pure powder sample of Cordyceps sinensis in Qinghai.
1, the instrument that adopts is measured and medicine 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 that Qinghai is good converts Cordyceps sinensis pure powder sample 0.25g, add water after 10ml and extract 30min in 60 DEG C of water-baths, after its cooling, add water and be settled to 50ml, become the worm summer grass product aqueous solution that concentration is 5.00mg/ml, filter this solution, get 2ml filtrate, add by centrifuge 15min after 6ml absolute ethyl alcohol, the rotating speed of hydro-extractor is 9000rpm, then taking precipitate adopt volume fraction be 5% methanol aqueous solution dissolve, to cross after 0.22 μm of organic filter membrane sample solution is for subsequent use;
(2) mensuration of high performance liquid chromatograph
Step (one) gained sample solution is got to join in high performance liquid chromatograph in right amount and measures, the liquid phase chromatogram condition adopted is: chromatographic column is Atlantis T3 chromatographic column, its specification is 4.6 × 250mm, i.d..5 μm, column temperature is 35 DEG C, mobile phase to be volume fraction be 5% methanol aqueous solution, 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 pipe detector enters injection device again after polychromatic light is absorbed by compositional selecting by sample cell, being radiated on diode array device makes the light intensity of each nano wave length be transformed into corresponding electrical signal intensity, namely obtains the absorption spectrum of component.Therefore, can be more preliminary qualitative to compound with standard substance retention time according to the chromatographic retention of object (namely occurring time during this concentration of component maximum value from sample introduction after post), utilize object more qualitative with the absorption spectrum of standard substance 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 is close, 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 determine that this component is Chinese caterpillar fungus polysaccharide peptide further; Like this dual qualitative more accurately and reliably, can more directly, more promptly to detect in sample whether containing 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, by water-soluble solution and constant volume, obtain the Chinese caterpillar fungus polysaccharide peptide standard reserve liquid that concentration is 5.00mg/ml, then 100 μ l are got respectively, 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 working solution of 500.0 μ g/ml, standard working solution is measured by high performance liquid chromatograph, as Fig. 5, the data obtained 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) determination of Chinese caterpillar fungus polysaccharide peptide content in sample
By the standard equation of the peak area of Chinese caterpillar fungus polysaccharide peptide measured in sample solution in step (two) for people's step (three) gained, be converted into the concentration of Chinese caterpillar fungus polysaccharide peptide, thus to calculate the good content converting Chinese caterpillar fungus polysaccharide peptide in the pure powder sample of Cordyceps sinensis in Qinghai be 16.8mg/g.
These are only better embodiment of the present invention, all equalizations done 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 (5)
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, water-bath extraction is carried out after adding water, after its cooling, the constant volume that adds water becomes concentration to be the worm summer grass product aqueous solution of 5.00mg/ml, filter this solution, getting portion filtrate adds by centrifuge after absolute ethyl alcohol, and then taking precipitate adopts mobile phase to dissolve, and to cross after organic filter membrane sample solution is for subsequent use;
(2) mensuration of high performance liquid chromatograph
Step (one) gained sample solution is got to join in high performance liquid chromatograph in right amount and measures, and qualitative analysis is carried out to measurement result;
(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, by water-soluble solution and constant volume, obtain the Chinese caterpillar fungus polysaccharide peptide standard reserve liquid that concentration is 5.00mg/ml, then 100 μ l are got respectively, 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 working solution of 500.0 μ g/ml, standard working solution is measured by high performance liquid chromatograph, the data obtained take concentration as horizontal ordinate, peak area is ordinate, set up concentration-peak area typical curve, and draw standard equation,
(4) determination of Chinese caterpillar fungus polysaccharide peptide content in sample
According to the linear equation that concentration-peak area typical curve draws, the peak area of Chinese caterpillar fungus polysaccharide peptide measured in sample solution in step (two) is scaled concentration, thus calculates the Chinese caterpillar fungus polysaccharide peptide content in this Cordyceps sinensis product;
When high performance liquid chromatograph measures, liquid phase chromatogram condition is: chromatographic column is Atlantis T3 chromatographic column, its specification is 4.6 × 250mm, i.d..5 μm, column temperature is 35 DEG C, mobile phase to be volume fraction be 5% methanol aqueous solution, flow velocity is 1ml/min, and sample size is 10 μ l, detecting device is diode array detector, and scanning wavelength is 190nm-460nm.
2. 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 DEG C, and the time is 30 minutes.
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 rotating speed of hydro-extractor used is 9000rpm, and centrifugation time is 15 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: organic filter membrane used is 0.22 μm of organic filter membrane.
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: described Cordyceps sinensis product comprises Cordyceps sinensis medicinal raw material and Chinese caterpillar fungus health-care food.
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| MX2017006443A (en) * | 2014-11-18 | 2017-09-11 | Vertex Pharma | Process of conducting high throughput testing high performance liquid chromatography. |
| 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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