CN103969384A - Content determination method of arca subcrenata hyperglycemia - Google Patents
Content determination method of arca subcrenata hyperglycemia Download PDFInfo
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- 201000001421 hyperglycemia Diseases 0.000 title abstract 4
- 238000002360 preparation method Methods 0.000 claims abstract description 22
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 4
- 238000002798 spectrophotometry method Methods 0.000 claims abstract description 3
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- 239000007788 liquid Substances 0.000 claims description 13
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- 238000003556 assay Methods 0.000 claims description 11
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- 238000002835 absorbance Methods 0.000 claims description 7
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 claims description 7
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 5
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Abstract
The invention relates to a content determination method of arca subcrenata hyperglycemia, which is characterized in that whether hyperglycemia in a to-be-measured sample is the arca subcrenata hyperglycemia is determined by using gel chromatography column high-performance liquid chromatography firstly, and then the content is determined by using a spectrophotometric method; and arca subcrenata polypeptide contained in an arca subcrenata extractive per gram in a preparation is not less than 24 mg, and when the content is higher, the curative effect is better. The content determination method can be used for determining the content of the arca subcrenata extractive in various preparations and has the benefits that the product quality is effectively ensured during the production and the use of the arca subcrenata preparation, and further, the curative effect of drugs is ensured.
Description
Technical field
The present invention relates to medicine detection method, be specifically related to a kind of content assaying method of blood clam polysaccharide, belong to field of pharmaceutical technology.
Background technology
Blood clam meat, containing rich in protein, vitamin and protein, polysaccharide, just has edible, medical value from ancient times, " medical center bun will " note " blood clam meat bushing blood, loose hemostasis, relieving restlessness are sobered up, the dissolving phlegm of broken knot ", " Japan hanako materia medica " note " blood clam meat benefit color ".In recent years, research report shows that blood clam has antitumor action, have effect of removing interior free yl, improving immunity of organisms, and security is good.
One of main bioactive ingredients of blood clam according to bibliographical information blood clam polysaccharide, have antitumor, regulate immune effect.Doctoral advisor professor Shen Yulong of China Medicine University report, obtains blood clam polysaccharide products from separation and purification in blood clam body, can promote significantly the propagation of splenic lymphocyte, has significant external immunocompetence.
Polysaccharide is the base substance of vital movement, all animals and plants life entities all contain polysaccharide, in the situation that cannot determining polysaccharide origin, just can not ensure the validity of medicine, content that can not Accurate Determining polysaccharide, just cannot in producing and using, ensure product quality, and then pharmaceutical effectiveness can not be guaranteed.
Existing document does not also have the report of blood clam polysaccharide specificity and assay.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of content assaying method of blood clam polysaccharide is provided, make blood clam preparation in producing and using, effectively ensure product quality.
A content assaying method for blood clam polysaccharide, is characterized in that first determining that by gel chromatographic columns high performance liquid chromatography the polysaccharide in sample is blood clam polysaccharide, then uses spectrophotometry content, and concrete steps are as follows:
(1) chromatographic condition and system flexibility: the gel chromatographic columns that is filling agent with hydrophilic spherical superpolymer; Taking the metabisulfite solution of 0.1mol/L as mobile phase; Differential refraction detector; Column temperature: 40 DEG C; Flow velocity: 0.5ml/min; Number of theoretical plate calculates and should be not less than 5000 by glucose peaks;
(2) take the sample containing the about 2g of blood clam extract, add the solution of the sherwood oil of 15 times: ethanol=1:1, in 40 DEG C of water bath heat preservation 3hr, ultrasonic extraction 30min, absorbent cotton filters, and gets filter residue, volatilizes; Add deionized water 20ml, with sodium hydroxide solution tune pH to 7.5～8,80 DEG C of water-bath 3hr, stir constantly, filter, the another device of filtrate is preserved, filter residue adds deionized water 20ml, continues water-bath 2hr, stirs constantly, filter, filtrate merges, more than adding 3 times of amount ethanol precipitation 6hr, abandon supernatant, subnatant is centrifugal, and precipitation adds deionized water 20ml, 80 DEG C of water-bath 2hr, centrifugal, get supernatant; Add the solution of the chloroform of 1/4 volume: normal butyl alcohol=4:1, violent jolting, centrifugal, get supernatant liquid, repetitive operation repeatedly, disappears completely to albumin layer in the middle of liquid level, gets supernatant liquid, more than adding 3 times of amount ethanol precipitation 6hr, abandon supernatant, subnatant is centrifugal, gets precipitation, 80 DEG C of following drying under reduced pressure, make blood clam polysaccharide.Get blood clam polysaccharide, add the mutual-assistance of flowing and dissolve, make the solution of every 1ml containing 10mg, to obtain final product;
(3) get blood clam polysaccharide need testing solution 20 μ l, injection liquid chromatography, measures and records collection of illustrative plates; If in collection of illustrative plates taking the S peak in collection of illustrative plates as reference, present 2 characteristic peaks, and taking S peak as 1, the relative retention time of 2 characteristic peaks at peak 1 is 0.56 ± 10%, peak 2 is 0.68 ± 10%, its weight-average molecular weight (Mw) is between 6.88～9.47 × 105, proceed following operation, as nothing is judged to adulterant;
(4) take containing the about 1-4g sample of blood clam extract, accurately weighed, put in round-bottomed flask, add 30-50 times of water, add hot reflux 1-3 hour, filter with absorbent cotton, filtrate moves in 50-150ml measuring bottle, add water to scale, shake up, precision measures 1-5ml, add 5-10 times of ethanol, stir, centrifugal 5-20 minute, precipitation is dissolved in water, put in 20-50ml measuring bottle, and be diluted to scale, shake up, precision measures 1-5ml, add 4% phenol solution of 1/2 volume, mix, add rapidly sulfuric acid 5-10ml, shake up, in 30-50 DEG C of water-bath, be incubated 20-60 minute, take out, put 2-10 minute in ice-water bath, take out, obtain blood clam polysaccharide test sample.Get blood clam polysaccharide need testing solution taking corresponding reagent as blank, according to UV-VIS spectrophotometry, measure absorbance at the wavelength place of 488nm.Read the weight (mg) of anhydrous dextrose in need testing solution from typical curve, calculate content.We are through repetition test discovery, and in preparation, every g blood clam extract, containing blood clam polysaccharide in glucose (C6H12O6), must not be less than 24mg, and content is higher, better efficacy.
The sample polyoses content of blood clam extract after vacuum drying is 13%, in blood clam preparation chemical constitution study, we find, the activity of polysaccharide with go relative molecular weight quality relevant, relative molecular mass is larger, molecular volume is larger, is unfavorable for polysaccharide performance biologically active, and the polysaccharide of little molecular weight is main active.And the stalwart blood clam of macromolecule, mud blood clam, flower clam, Scallop Extract, its contained polysaccharide molecular weight is different from blood clam polysaccharide, use gel chromatographic columns high effective liquid chromatography for measuring, in relative retention time 0.56 ± 10% and 0.68 ± 10% without blood clam polysaccharide characteristic peak, therefore strong by the content specificity of method mensuration blood clam polysaccharide of the present invention, can ensure that the medicine of preparing with blood clam is effectively with quality controllable.
For the ease of understanding the present invention, special further illustrate by test example below:
One, two batches of blood clam extracts
(1) chromatographic condition and system flexibility: the gel chromatographic columns that is filling agent with hydrophilic spherical superpolymer; Taking the metabisulfite solution of 0.1mol/L as mobile phase; Differential refraction detector; Column temperature: 40 DEG C; Flow velocity: 0.5ml/min; Number of theoretical plate calculates and should be not less than 5000 by glucose peaks;
(2) take containing the each 2g of blood clam extract, add respectively the solution of the sherwood oil of 15 times: ethanol=1:1, in 40 DEG C of water bath heat preservation 3hr, ultrasonic extraction 30min, absorbent cotton filters, and gets filter residue, volatilizes; Add deionized water 20ml, with sodium hydroxide solution tune pH to 7.5～8,80 DEG C of water-bath 3hr, stir constantly, filter, the another device of filtrate is preserved, filter residue adds deionized water 20ml, continues water-bath 2hr, stirs constantly, filter, filtrate merges, more than adding 3 times of amount ethanol precipitation 6hr, abandon supernatant, subnatant is centrifugal, and precipitation adds deionized water 20ml, 80 DEG C of water-bath 2hr, centrifugal, get supernatant; Add the solution of the chloroform of 1/4 volume: normal butyl alcohol=4:1, violent jolting, centrifugal, get supernatant liquid, repetitive operation repeatedly, disappears completely to albumin layer in the middle of liquid level, gets supernatant liquid, more than adding 3 times of amount ethanol precipitation 6hr, abandon supernatant, subnatant is centrifugal, gets precipitation, 80 DEG C of following drying under reduced pressure, make blood clam polysaccharide.Get blood clam polysaccharide, add the mutual-assistance of flowing and dissolve, make the solution of every 1ml containing 10mg, to obtain final product;
(3) get the each 20 μ l of blood clam polysaccharide need testing solution, injection liquid chromatography, measures and records collection of illustrative plates; In A sample characteristic collection of illustrative plates, present 2 characteristic peaks, taking the S peak in collection of illustrative plates as 1, peak 1 relative retention time is 0.58, peak 2 relative retention times are 0.69, its molecular weight is 688036, in B sample characteristic collection of illustrative plates, present 2 characteristic peaks, taking the S peak in collection of illustrative plates as 1, peak 1 relative retention time is 0.55, peak 2 relative retention times are 0.63.Its molecular weight is 784503, and result shows that the agent of two batches of blood clam extract particles is all to make with blood clam.Proceed assay;
(4) take respectively the sample containing blood clam extract 2.5g, accurately weighed, put in round-bottomed flask, add 40 times of water, add hot reflux 2 hours, filter with absorbent cotton, filtrate moves in 50-150ml measuring bottle, add water to scale, shake up, precision measures 2ml, add 9 times of ethanol, stir, centrifugal 10 minutes, precipitation is dissolved in water, put in 50ml measuring bottle, and be diluted to scale, shake up, precision measures 2ml, add 4% phenol solution of 1/2 volume, mix, add rapidly sulfuric acid 7ml, shake up, in 40 DEG C of water-baths, be incubated 30 minutes, take out, put in ice-water bath 5 minutes, take out, obtain blood clam polysaccharide test sample.Get blood clam polysaccharide need testing solution taking corresponding reagent as blank, according to UV-VIS spectrophotometry, measure absorbance at the wavelength place of 488nm.Read the weight (mg) of anhydrous dextrose in need testing solution from typical curve, calculate content.
In preparation A sample, every g blood clam extract is containing polysaccharide 53mg.In preparation B sample, every g blood clam extract is containing polysaccharide 28mg.
Two, two kinds of preparation drug effects comparison: blood clam extract formulation immunoregulation effect test
Test method: choose 30 of healthy mices, be divided at random 3 groups, each group all gives preparation A, B 0.25g/kg intravenous injection, blank group injection equivalent physiological saline.Each group all once a day, continuous 15 days.
Observation item: after last administration, mouse is put to death in cervical vertebra dislocation, weighs, and taking-up mouse spleen, thymus gland are weighed, and calculated organ index.
The results are shown in Table:
| Group | Body weight/g | Thymus index | Spleen index |
| Preparation A | 20.91±0.84 | 4.43±0.41 | 5.71±0.43 |
| Preparation B | 20.25±0.95 | 4.01±0.43 | 5.24±0.40 |
| Blank | 20.64±0.77 | 3.42±0.38 | 4.56±0.36 |
Feed after 15d, blood clam extract has no significant effect the body weight of mouse, and preparation A is all being better than preparation B aspect thymus index, spleen index, and has significant difference.Show that preparation A curative effect is better than preparation B.
Below in conjunction with embodiment, the invention will be further described.
Embodiment 1
Get fresh blood clam meat, fragmentation, crosses 40 mesh sieves, adds 1 times of water gaging, extract 4 hours 10 DEG C of following stirrings, and extracting liquid filtering, centrifugal, get above-mentioned centrifugate freeze-drying, obtain blood clam extract.
Blood clam extract characteristic spectrum presents 2 characteristic peaks, and taking the S peak in collection of illustrative plates as 1, peak 1 relative retention time is 0.55, peak 2 relative retention times are 0.62, and its molecular weight is 758050.Result shows that blood clam extract is to make with blood clam.Proceed assay.
Get blood clam extract porphyrize, precision takes 1.028g, put in round-bottomed flask, add 30 times of water, add hot reflux 2 hours, filter with absorbent cotton, filtrate moves in 100ml measuring bottle, add water to scale, shake up, precision measures 3ml, add 7 times of ethanol, stir, centrifugal 30 minutes, precipitation is dissolved in water, put in 25ml measuring bottle, and be diluted to scale, shake up, precision measures 2ml, add 4% phenol solution of 1/2 volume, mix, add rapidly sulfuric acid 8ml, shake up, in 30 DEG C of water-baths, be incubated 20 minutes, take out, put in ice-water bath 2 minutes, take out, obtain blood clam polysaccharide test sample.Get blood clam polysaccharide need testing solution taking corresponding reagent as blank, according to UV-VIS spectrophotometry, measure absorbance at the wavelength place of 488nm.Read the weight (mg) of anhydrous dextrose in need testing solution from typical curve, calculate content.In preparation A sample, every g blood clam extract is containing polysaccharide 61mg.
Embodiment 2
Get dry blood clam meat, pulverize, cross 80 mesh sieves, add the water of 10 times of amounts, heating is extracted 2 times, and each 1 hour, merge extract, filter, get filtrate, being concentrated into relative density is 1.04～1.06(60 DEG C) time, centrifugal.Get above-mentioned centrifugate below 80 DEG C, drying under reduced pressure, pulverizes, and obtains blood clam extract.Extract is made to tablet.
Blood clam extract characteristic spectrum presents 2 characteristic peaks, and taking the S peak in collection of illustrative plates as 1, peak 1 relative retention time is 0.58, peak 2 relative retention times are 0.72, and its molecular weight is 693650.Result shows that blood clam extract is to make with blood clam.Proceed assay.
Get blood clam extract tablet porphyrize, precision takes 3.764g, containing blood clam extract 3.011g, put in round-bottomed flask, add 40 times of water, add hot reflux 1 hour, filter with absorbent cotton, filtrate moves in 50ml measuring bottle, add water to scale, shake up, precision measures 1ml, add 5 times of ethanol, stir, centrifugal 10 minutes, precipitation is dissolved in water, put in 20ml measuring bottle, and be diluted to scale, shake up, precision measures 5ml, add 4% phenol solution of 1/2 volume, mix, add rapidly sulfuric acid 5ml, shake up, in 40 DEG C of water-baths, be incubated 40 minutes, take out, put in ice-water bath 50 minutes, take out, obtain blood clam polysaccharide test sample.Get blood clam polysaccharide need testing solution taking corresponding reagent as blank, according to UV-VIS spectrophotometry, measure absorbance at the wavelength place of 488nm.Read the weight (mg) of anhydrous dextrose in need testing solution from typical curve, calculate content.In preparation A sample, every g blood clam extract is containing polysaccharide 41mg.
Embodiment 3
Prepare blood clam extract according to the blood clam method for preparing extractive in embodiment 2, extract is made to capsule.
Get capsule 's content and differentiate, in result reality blood clam extract characteristic spectrum, present 2 characteristic peaks, taking the S peak in collection of illustrative plates as 1, peak 1 relative retention time is 0.51, peak 2 relative retention times are 0.62, and its molecular weight is 796320.Result shows that blood clam extract is to make with blood clam.Proceed assay.
Get capsule 's content porphyrize, precision takes 5.359g, containing blood clam extract 4.823g, put in round-bottomed flask, add 50 times of water, add hot reflux 3 hours, filter with absorbent cotton, filtrate moves in 150ml measuring bottle, add water to scale, shake up, precision measures 5ml, add 10 times of ethanol, stir, centrifugal 20 minutes, precipitation is dissolved in water, put in 50ml measuring bottle, and be diluted to scale, shake up, precision measures 1ml, add 4% phenol solution of 1/2 volume, mix, add rapidly sulfuric acid 10ml, shake up, in 50 DEG C of water-baths, be incubated 60 minutes, take out, put in ice-water bath 10 minutes, take out, obtain blood clam polysaccharide test sample.Get blood clam polysaccharide need testing solution taking corresponding reagent as blank, according to UV-VIS spectrophotometry, measure absorbance at the wavelength place of 488nm.Read the weight (mg) of anhydrous dextrose in need testing solution from typical curve, calculate content.In preparation A sample, every g blood clam extract is containing polysaccharide 84mg.
Embodiment 4
According to the blood clam method for preparing extractive index blood clam extract in embodiment 2, extract is prepared into granule.
Get granule content and differentiate, concrete steps are as follows:
Chromatographic condition and system flexibility: the gel chromatographic columns that is filling agent with hydrophilic spherical superpolymer; Taking the metabisulfite solution of 0.1mol/L as mobile phase; Differential refraction detector; Column temperature: 40 DEG C; Flow velocity: 0.5ml/min; Number of theoretical plate calculates and should be not less than 5000 by glucose peaks.
Take containing blood clam extract particles 2g, add respectively the solution of the sherwood oil of 15 times: ethanol=1:1, in 40 DEG C of water bath heat preservation 3hr, ultrasonic extraction 30min, absorbent cotton filters, and gets filter residue, volatilizes; Add deionized water 20ml, with sodium hydroxide solution tune pH to 7.5～8,80 DEG C of water-bath 3hr, stir constantly, filter, the another device of filtrate is preserved, filter residue adds deionized water 20ml, continues water-bath 2hr, stirs constantly, filter, filtrate merges, more than adding 3 times of amount ethanol precipitation 6hr, abandon supernatant, subnatant is centrifugal, and precipitation adds deionized water 20ml, 80 DEG C of water-bath 2hr, centrifugal, get supernatant; Add the solution of the chloroform of 1/4 volume: normal butyl alcohol=4:1, violent jolting, centrifugal, get supernatant liquid, repetitive operation repeatedly, disappears completely to albumin layer in the middle of liquid level, gets supernatant liquid, more than adding 3 times of amount ethanol precipitation 6hr, abandon supernatant, subnatant is centrifugal, gets precipitation, 80 DEG C of following drying under reduced pressure, make blood clam polysaccharide.Get blood clam polysaccharide, add the mutual-assistance of flowing and dissolve, make the solution of every 1ml containing 10mg, to obtain final product.
Get the each 20 μ l of blood clam polysaccharide need testing solution, injection liquid chromatography, measures and records collection of illustrative plates; In collection of illustrative plates, present 2 characteristic peaks, taking the S peak in collection of illustrative plates as 1, peak 1 relative retention time is 0.52, peak 2 relative retention times are 0.62, and its molecular weight is 788156, and result shows that the agent of blood clam extract particles is to make with blood clam.Proceed assay;
Get blood clam extract particles porphyrize, precision takes 2.802g, containing blood clam extract 2.522g, put in round-bottomed flask, add 25 times of water, add hot reflux 2 hours, filter with absorbent cotton, filtrate moves in 50ml measuring bottle, add water to scale, shake up, precision measures 2ml, add 10 times of ethanol, stir, centrifugal 15 minutes, precipitation is dissolved in water, put in 25 measuring bottles, and be diluted to scale, shake up, precision measures 0ml, add 4% phenol solution of 1/2 volume, mix, add rapidly sulfuric acid 8ml, shake up, in 50 DEG C of water-baths, be incubated 30 minutes, take out, put in ice-water bath 80 minutes, take out, obtain blood clam polysaccharide test sample.Get blood clam polysaccharide need testing solution taking corresponding reagent as blank, according to UV-VIS spectrophotometry, measure absorbance at the wavelength place of 488nm.Read the weight (mg) of anhydrous dextrose in need testing solution from typical curve, calculate content.In preparation A sample, every g blood clam extract is containing polysaccharide 66g.
Embodiment 5
Get respectively the blood clam in the different places of production, prepare 3 batches of blood clam extract pills by the method for embodiment 2, extract respectively sample A, sample B and the sample C of three crowdes, differentiate and assay.3 batches of measurement results are:
In sample A characteristic spectrum, present 2 characteristic peaks, taking the S peak in collection of illustrative plates as 1, peak 1 relative retention time is 0.52, peak 2 relative retention times are 0.60, and its molecular weight is 732520.Result shows that blood clam extract is to make with blood clam.Proceed assay.The every g of blood clam extract is containing blood clam polysaccharide 29mg.
In sample B characteristic spectrum, present 2 characteristic peaks, taking the S peak in collection of illustrative plates as 1, peak 1 relative retention time is 0.57, peak 2 relative retention times are 0.66, and its molecular weight is 831654.Result shows that blood clam extract is to make with blood clam.Proceed assay.The every g of blood clam extract is containing blood clam polysaccharide 34mg.
In sample A characteristic spectrum, present 2 characteristic peaks, taking the S peak in collection of illustrative plates as 1, peak 1 relative retention time is 0.55, peak 2 relative retention times are 0.63, and its molecular weight is 856280.Result shows that blood clam extract is to make with blood clam.Proceed assay.The every g of blood clam extract is containing blood clam polysaccharide 54mg.
Claims (5)
1. a content assaying method for blood clam polysaccharide, is characterized in that first determining that by gel chromatographic columns high performance liquid chromatography the polysaccharide in sample is blood clam polysaccharide, then uses spectrophotometry content.
2. determine that according to the gel chromatographic columns high performance liquid chromatography of claim 1 polysaccharide in sample is blood clam polysaccharide, it is characterized in that concrete steps are as follows:
(1) chromatographic condition and system flexibility: the gel chromatographic columns that is filling agent with hydrophilic spherical superpolymer; Taking the metabisulfite solution of 0.1mol/L as mobile phase; Differential refraction detector; Column temperature: 40 DEG C; Flow velocity: 0.5ml/min; Number of theoretical plate calculates and should be not less than 5000 by glucose peaks;
(2) take the sample containing the about 2g of blood clam extract, add the solution of the sherwood oil of 15 times: ethanol=1:1, in 40 DEG C of water bath heat preservation 3hr, ultrasonic extraction 30min, absorbent cotton filters, and gets filter residue, volatilizes; Add deionized water 20ml, with sodium hydroxide solution tune pH to 7.5～8,80 DEG C of water-bath 3hr, stir constantly, filter, the another device of filtrate is preserved, filter residue adds deionized water 20ml, continues water-bath 2hr, stirs constantly, filter, filtrate merges, more than adding 3 times of amount ethanol precipitation 6hr, abandon supernatant, subnatant is centrifugal, and precipitation adds deionized water 20ml, 80 DEG C of water-bath 2hr, centrifugal, get supernatant; Add the solution of the chloroform of 1/4 volume: normal butyl alcohol=4:1, violent jolting, centrifugal, get supernatant liquid, repetitive operation repeatedly, disappears completely to albumin layer in the middle of liquid level, gets supernatant liquid, more than adding 3 times of amount ethanol precipitation 6hr, abandon supernatant, subnatant is centrifugal, gets precipitation, 80 DEG C of following drying under reduced pressure, make blood clam polysaccharide; Get blood clam polysaccharide, add the mutual-assistance of flowing and dissolve, make the solution of every 1ml containing 10mg;
(3) get blood clam polysaccharide need testing solution 20 μ l, injection liquid chromatography, measures and records collection of illustrative plates; If in collection of illustrative plates taking the S peak in collection of illustrative plates as reference, present 2 characteristic peaks, and taking S peak as 1, the relative retention time of 2 characteristic peaks at peak 1 is 0.56 ± 10%, peak 2 is 0.68 ± 10%, its weight-average molecular weight (Mw) is between 6.88～9.47 × 105, proceed following operation, as nothing is judged to adulterant.
3. according to the content assaying method of the blood clam polysaccharide of claim 1, it is characterized in that taking containing the about 1-4g sample of blood clam extract, accurately weighed, put in round-bottomed flask, add 30-50 times of water, add hot reflux 1-3 hour, filter with absorbent cotton, filtrate moves in 50-150ml measuring bottle, add water to scale, shake up, precision measures 1-5ml, add 5-10 times of ethanol, stir, centrifugal 5-20 minute, precipitation is dissolved in water, put in 20-50ml measuring bottle, and be diluted to scale, shake up, precision measures 1-5ml, add 4% phenol solution of 1/2 volume, mix, add rapidly sulfuric acid 5-10ml, shake up, in 30-50 DEG C of water-bath, be incubated 20-60 minute, take out, put 2-10 minute in ice-water bath, take out, obtain blood clam polysaccharide test sample, get blood clam polysaccharide need testing solution taking corresponding reagent as blank, according to UV-VIS spectrophotometry, measure absorbance at the wavelength place of 488nm, read the weight (mg) of anhydrous dextrose in need testing solution from typical curve, calculate content.
4. according to the content assaying method of a kind of blood clam polysaccharide of claim 1 or claim 3, it is characterized in that in preparation, every g blood clam extract must not be less than 24mg containing blood clam polysaccharide, content is higher, better efficacy.
5. according to the content assaying method of a kind of blood clam polysaccharide of claim 1 or claim 2 or claim 3 or claim 4, it is characterized in that can be used for the assay of the various preparations of blood clam extract.
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