CN103592382B - A kind of method detecting and determine sulfate radical covalently bound in Cordyceps sinensis polysaccharide - Google Patents
A kind of method detecting and determine sulfate radical covalently bound in Cordyceps sinensis polysaccharide Download PDFInfo
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- CN103592382B CN103592382B CN201310538376.3A CN201310538376A CN103592382B CN 103592382 B CN103592382 B CN 103592382B CN 201310538376 A CN201310538376 A CN 201310538376A CN 103592382 B CN103592382 B CN 103592382B
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Abstract
The invention belongs to field of medicaments, relate to a kind of method detecting and determine sulfate radical covalently bound in Cordyceps sinensis polysaccharide, detecting step comprises (1) by Na
2 34sO
4add fluid nutrient medium; (2) Chinese caterpillar fungus seed liquor is added in the nutrient culture media of step (1) and cultivate; (3) from the Cordyceps Militaris of step (2) gained, Cordyceps sinensis polysaccharide is extracted; (4) Cordyceps sinensis polysaccharide extracted in step (3) is carried out intermediate acid degraded, and mark catabolite with aniline; (5) sulfate radical covalently bound with sugar in the product obtained by liquid chromatography mass spectrometric coupling method detecting step (4).The Na added in nutrient culture media
2 34sO
4concentration be 1-1000ug/mL.The method amount of samples is few, highly sensitive, use safety, and the characterization of biological activity for Cordyceps sinensis polysaccharide provides a New Set.
Description
Technical field
The invention belongs to field of medicaments, relate to a kind of method detecting and determine sulfate radical covalently bound in Cordyceps sinensis polysaccharide.
Background technology
Cordyceps sinensis (Cordyceps), has another name called Chinese caterpillar fungus, CORDYCEPS, Cordyceps sinensis.Belong to mycota, Mycophyta, Ascomycetes, Hypocreales, Clavicipitaceae, Cordyceps.Be a kind ofly colonize in the entomogenous fungi complex that bat moth larvae is formed, be mainly distributed in the high altitude localities of China Qinghai, Tibet, Sichuan, Yunnan, Guizhou, Gansu height above sea level about 4000 meters.Natural cs growing environment is special, and price is higher, and people cultivate cordyceps mycelia with biofermentation technique and carry out alternative Cordyceps sinensis, and its composition, medical value and Cordyceps sinensis are similar.Cordyceps sinensis polysaccharide is the main active of Chinese caterpillar fungus, comprises as anti-inflammatory, anti-oxidant, resisiting influenza virus, and immunological regulation is hypoglycemic, antitumor and Anti-angiogenesis isoreactivity.These are active relevant with the structure of Cordyceps sinensis polysaccharide.
Sulfated polysaccharide has diversified biologically active, comprises anti-freezing, and anti-inflammatory is anti-oxidant, antiviral, hypoglycemic, antitumor and Anti-angiogenesis isoreactivity.Heparin as terraria source applies 78 years clinically as anticoagulation, and the derivant PSS of polysaccharides of marine algae also also applies 27 years in China as the medicine of ischemic cerebrovascular disease clinically.Sulfated polysaccharide is the important biomolecule active component of sea-plant, marine animal, terraria cell and tissue.Our Late Cambrian, the Cordyceps sinensis polysaccharide in Cordyceps sinensis contains covalently bound sulfate radical.
The detection method of sulfated polysaccharide sulfate radical conventional at present has elemental microanalysis method, barium sulphate gelatin turbidimetry etc.A lot of shortcoming is there is in these methods in the detection of sulfate radical, as elemental microanalysis method is very high to the purity requirement of sample, resolving ability is not had for impurity, result can only illustrate that element sulphur is present in sample, but can not be explained for element sulphur existence form in the sample to which (organic sulfur, inorganic sulfur).Barium sulphate gelatin turbidimetry consumption sample amount is large, and detect limit for height, the repeatability of result is bad.These two kinds of methods all can not the interference of despumation, can not determine that element sulphur derives from polysaccharide actually, nutrient culture media or protein, and sample consumption is large.At present not yet relevant Cordyceps sinensis polysaccharide contains the report of covalently bound sulfate radical, exactly because the restriction of this detection method causes.
Summary of the invention
The present invention relates to a kind of method detecting and determine sulfate radical covalently bound in Cordyceps sinensis polysaccharide, the method amount of samples is few, detection sensitivity is high, the isotope used is "dead", use safety, can detect sulfate radical covalently bound in Cordyceps sinensis polysaccharide qualitatively, not by nutrient culture media, the interference of the impurity such as protein, just can detect without the need to standard items.For the detection of Cordyceps sinensis polysaccharide and the covalently bound sulfate radical of other microbial polysaccharides provides method, simultaneously for the characterization of biological activity of Cordyceps sinensis polysaccharide provides a New Set.
The technical method realizing foregoing invention object is:
1. detect and determine a method for sulfate radical covalently bound in Cordyceps sinensis polysaccharide, specifically comprise the following steps:
(1) by Na
2 34sO
4add fluid nutrient medium;
(2) Chinese caterpillar fungus seed liquor is added in the nutrient culture media of step (1) and cultivate;
(3) from the Cordyceps Militaris of step (2) gained, Cordyceps sinensis polysaccharide is extracted;
(4) Cordyceps sinensis polysaccharide extracted in step (3) is carried out intermediate acid degraded, and mark catabolite with aniline;
(5) sulfate radical covalently bound with sugar in the product obtained by liquid chromatography mass spectrometric coupling method detecting step (4).
2. the Na added in fluid nutrient medium
2 34sO
4concentration be 1-1000ug/mL.
Compared with prior art, the present invention has following Advantageous Effects:
(1) the method amount of samples is few, can not cause the waste of sample.
(2) detection sensitivity is high, in conjunction with liquid chromatography mass spectrometric coupling method, even if the sulfate radical containing trace in sample also can detect, can also determine that sulfate radical exists with inorganic or organic form.
(3) isotope used is "dead", can use safely.
(4) interference not by impurity in testing process.
(5) just can detect without the need to standard items.
(6) be not only applicable to Cordyceps Militaris, be also applicable to the detection of sulfate radical covalently bound in other microbial polysaccharides.
(7) 2 kinds of samples can be detected simultaneously, and relative quantification is carried out to these two kinds of samples.
Accompanying drawing explanation
Fig. 1 is the aniline marked product mass spectrogram of embodiment 3 Sulfated saccharides
Fig. 2 is the deuterated aniline marked product mass spectrogram of embodiment 2 Sulfated saccharides
Embodiment
The Paecilomyces hepiali chen (Paecilomyces hepiali) that following examples adopt, bacterium numbering 3.7845, purchased from China Committee for Culture Collection of Microorganisms's common micro-organisms center.
Embodiment 1
Bacterial strain increases: with PDA solid medium, Paecilomyces hepiali chen is carried out bacterial strain amplification, cultivates 7 days under 27 DEG C of conditions;
Seed liquor is cultivated: from solid medium, picking mycelium is in 150mL potato fluid nutrient medium, puts 180r/min on shaking table, cultivates 10 days under 27 DEG C of conditions;
Get 6mL seed liquor to add containing 1ug/mL Na
2 34sO
4200mL potato liquid support in base, under 27 DEG C of conditions cultivate 10 days.
Embodiment 2
Bacterial strain amplification and seed liquor incubation step are with embodiment 1;
Get 6mL seed liquor to add containing 1000ug/mLNa
2 34sO
4200mL potato liquid support in base, under 27 DEG C of conditions cultivate 10 days.
Embodiment 3
Bacterial strain amplification and seed liquor incubation step are with embodiment 1;
Get 6mL seed liquor to add not containing Na
2 34sO
4200mL potato liquid support in base, under 27 DEG C of conditions cultivate 10 days.
The mycelium that embodiment 1-3 turns out extracts Cordyceps sinensis polysaccharide according to following method:
Nutrient solution is concentrated into 1/4 volume, adding absolute ethyl alcohol to the final concentration of ethanol is 80%, 80 DEG C of backflow 3h degreasings.Centrifugal 10min after cooling, gets precipitation, three times repeatedly, obtains degreasing product.Extracting degreasing product 2g, adds 40mL water, magnetic agitation 3h, centrifugal 10min, three times repeatedly.Supernatant is dialysed, then dislysate is concentrated, add 4 times of absolute ethyl alcohol alcohol precipitations, obtain Cordyceps sinensis polysaccharide.
Test a Cordyceps sinensis polysaccharide and carry out intermediate acid degraded and aniline mark, product detects by liquid chromatography mass spectrometric coupling (LC-MS) method and analyzes
Get 0.1mg embodiment 1-3 extract Cordyceps sinensis polysaccharide and 500uL0.05M trifluoroacetic acid in ampoule bottle, 1.5h is reacted under 100 DEG C of conditions, revolve after having reacted and steam to dry, in product, add the deuterated aniline of 10uL (embodiment 3 adds the common aniline of 10uL) and 10uL1M NaCNBH
3, under 65 DEG C of conditions, react 1h, product revolves and steams to dry.With 25uL distilled water dissolving and reducing aminate, 13000r/min is centrifugal, gets supernatant and carries out LC-MS analysis.Liquid-phase condition: liquid phase post 0.3 × 250mm SB-C18 post (5um, Agilent), flow velocity 5uL/min, mobile phase A contains 5% methanol aqueous solution of 3.5mM dibutylamine, Mobile phase B contains 95% methanol aqueous solution of 3.5mM dibutylamine, and applied sample amount is 0.1uL, mobile phase condition: mobile phase 0%B7min, 15%B9min, 40%B11min, 100%B23min, 232nm detect, mass spectrum is the lowest detectable limit 50ng of electrospray ionization mass spectrum negative ion mode, the method.The results are shown in Figure 1, Fig. 2.
M/z336.10 and m/z498.15 as can see from Figure 1, but do not add Na due to embodiment 3
2 34sO
4, therefore can not determine that whether these two mass numbers are the mass numbers of sulfated sugar, is likely the mass number of other impurity such as amino acid or phosphoric acid.As seen from Figure 2, each mass number is than the corresponding mass number of Fig. 1 large 5.03, this is because the deuterated aniline of embodiment 2 marks, m/z343.13 and m/z505.19 contains
34sO
4 2-sugar, be because bacterial classification make use of Na in nutrient culture media in growth course
2 34sO
4carry out the synthesis of sulfate radical on polysaccharide, these two mass numbers further illustrate the mass number that m/z341.13 and m/z503.18 is also sulfated sugar in polysaccharide, because bacterial classification also uses part in growth course
32s carries out the synthesis of sulfate radical on polysaccharide.So just can determine in Cordyceps sinensis polysaccharide containing covalently bound sulfate radical.
Test two barium sulfate turbidimetries and detect sulfate radical covalently bound in Cordyceps sinensis polysaccharide
Accurate configuration sulfate concentration is the K of 0.6mg/mL
2sO
4solution.Get 0,40,60,80,100,120uL standard solution, mend to 200uL with 1moL/L hydrochloric acid, add trichloroacetic acid 3.8mL and barium chloride gelatin solution 1mL, shake up, room temperature is placed 15min, 360nm and is surveyed absorbance.With sulfate concentration for horizontal ordinate, absorbance is ordinate drawing standard curve.Obtain equation of linear regression: y=0.0028x+0.0116 R
2=0.9979.
Precision takes 5mg sample and is placed in ampoule bottle, 2mL1M dissolving with hydrochloric acid, and sealing, at 110 DEG C of Water Under solution 6h.Get 100uL hydrolyzate, mend to 200uL with 1M hydrochloric acid, add trichloroacetic acid 3.8mL, barium chloride gelatin solution 1mL, shakes up, puts 15min in ambient temperatare, and 360nm measures absorbance log.Example 1,2,3 is analyzed, the results are shown in Table 1.
Table 1 barium sulphate gelatin turbidimetry detects sulfate radical result
As shown in Table 1, the sulfate radical of trace in Cordyceps sinensis polysaccharide cannot be detected with barium sulfate turbidimetry.The method sample consumption is large, compared with the sample size of the inventive method consumption, is 50 times of inventive samples consumption.
Claims (2)
1. detect and determine a method for sulfate radical covalently bound in Cordyceps sinensis polysaccharide, it is characterized in that, specifically comprise the following steps:
(1) by Na
2 34sO
4add fluid nutrient medium;
(2) Chinese caterpillar fungus seed liquor is added in the nutrient culture media of step (1) and cultivate;
(3) from the Cordyceps Militaris of step (2) gained, Cordyceps sinensis polysaccharide is extracted;
(4) Cordyceps sinensis polysaccharide extracted in step (3) is carried out intermediate acid degraded, and mark catabolite with aniline;
(5) sulfate radical covalently bound with sugar in the product obtained by liquid chromatography mass spectrometric coupling method detecting step (4);
Liquid-phase condition: 5 μm of Agilent liquid phase post 0.3 × 250mmSB-C18 posts, flow velocity 5 μ L/min, mobile phase A contains 5% methanol aqueous solution of 3.5mM dibutylamine, and Mobile phase B contains 95% methanol aqueous solution of 3.5mM dibutylamine, and applied sample amount is 0.1 μ L, mobile phase condition: mobile phase 0%B 7min, 15%B 9min, 40%B 11min, 100%B 23min, 232nm detects, and mass spectrum is electrospray ionization mass spectrum negative ion mode.
2. a kind of method detecting and determine sulfate radical covalently bound in Cordyceps sinensis polysaccharide according to claim 1, is characterized in that, the Na added in fluid nutrient medium
2 34sO
4concentration be 1-1000 μ g/mL.
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