CN103969366B - A kind of content assaying method of blood clam - Google Patents
A kind of content assaying method of blood clam Download PDFInfo
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Abstract
The present invention relates to the content assaying method of a kind of blood clam polypeptide and polysaccharide, it is characterized in that first with being blood clam in gel chromatographic columns high performance liquid chromatography determination sample, then measure the content of blood clam polypeptide and polysaccharide.The content of blood clam polypeptide is better effects if within the scope of 5-7 times of blood clam polyoses content.Make blood clam preparation effectively ensure product quality in production with in using, and then ensure pharmaceutical effectiveness.
Description
Technical field
The present invention relates to medicine detection method, be specifically related to a kind of content assaying method of blood clam, belong to field of pharmaceutical technology.
Background technology
Blood clam meat is containing the necessary amino acid of rich in protein, vitamin and human body, just there is 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 display blood clam has antitumor action, has the effect removed interior free yl, improve immunity of organisms.Blood clam meat preparation has good therapeutic action to kinds cancers such as lung cancer, kidney, cancer of the stomach, liver cancer, and security is good.
According to the primary bioactive components that bibliographical information blood clam polypeptide and polysaccharide are blood clams, have protect the liver, effect of antitumor, immunity moderation.Ji'nan University doctoral advisor Yu Rongmin teaches report on January 5th, 2011, fresh blood clam shells, through homogenate, centrifugal, dialysis, obtained blood clam polypeptide extract after freeze-drying, method measures the impact of mice spleen lymphocytes proliferation and the impact on mouse NK cell killing activity; On the impact of Phagocytosis By The Peritoneal Macrophages In Mice; The change in mouse spleen lymphocyte cycle; On the impact of major cytokine IFN-γ and IL-4 secretion level.Result blood clam polypeptide extract significantly can promote mice spleen lymphocytes proliferation, strengthens Turnover of Mouse Peritoneal Macrophages and engulfs dimethyl diaminophenazine chloride activity and mouse NK cell killing activity; Promote that splenic lymphocyte G0/G1 phase to DNA synthesis phase (s phase) transforms; Collaborative Con A effect, can increase the secretion of IFN-γ, and can suppress the secretion of IL-4.China Medicine University Dou Chang is expensive waits report; with blood clam meat hydrolyzate mouse stomach; result: all have obvious reducing effect to the serum glutamic pyruvic transminase activity rising that phenixin, thio-ethylamine and prednisolone cause, show that blood clam hydrolyzate has significant protective effect to Experimental Hepatic Damage.
We test and find that blood clam polypeptide obviously can suppress the expression of hepatitis B surface antigen and E antigen, and have inhibiting effect to hepatitis B DNA.
Protein and polysaccharide are the base substances of vital movement, all animals and plants life entities all contain this two kinds of base substances, when source cannot be determined, just can not ensure the validity of medicine, can not the content of accurate protein determination matter and polysaccharide, just cannot ensure product quality in production with in using, and then pharmaceutical effectiveness can not be guaranteed.
Existing document does not also have the report of blood clam polypeptide and polysaccharide specificity and assay.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, the content assaying method of a kind of blood clam polypeptide and polysaccharide is provided, make blood clam preparation effectively ensure product quality in production with in using.
A kind of content assaying method of blood clam, it is characterized in that, first by the polypeptide in gel chromatographic columns high performance liquid chromatography discriminating sample and polysaccharide, if two specificities are all satisfied, then determining it is blood clam, measure the content of polypeptide and polysaccharide in blood clam again, concrete steps are as follows:
(1) blood clam polypeptide is differentiated:
Chromatographic condition and system flexibility: be the gel chromatographic columns of filling agent with hydrophilic spherical superpolymer; With the phosphate buffer of 0.05mol/L for mobile phase; Determined wavelength is 280 nm; Column temperature: 25 DEG C; Flow velocity 0.4ml/min; Determined wavelength 280nm; Number of theoretical plate calculates by protein molecular weight 100000 and is not less than 5000;
Take sample 10g, add 12 times of water ultrasonic extraction 30 min, centrifugal 10 min, get upper liquid, filter, filtrate is mark 60%(w/v by volume) add ammonium sulfate, 0 ~ 4 DEG C of placement is spent the night, centrifugal, gets precipitation, the 10ml that adds water dissolves, and the ammonium bicarbonate soln with 1% is dislysate, is the dialysis membrane of 3500 with molecular cut off, enough hemodialysis under 0 ~ 4 DEG C of condition, is transferred to solution in dialysis membrane in 50ml measuring bottle, uses water constant volume, 0 ~ 4 DEG C of preservation, obtains blood clam polypeptide solution;
Get blood clam polypeptide solution 10 μ l, injection liquid chromatography, measure, record collection of illustrative plates; If there is the characteristic peak of blood clam polypeptide in collection of illustrative plates within retention time 15.6min ± 5%, its molecular weight is 88070, meets blood clam polypeptide specificity, then proceeds following operation, as nothing is then judged to adulterant;
(2) blood clam polysaccharide is differentiated:
Chromatographic condition and system flexibility: be the gel chromatographic columns of filling agent with hydrophilic spherical superpolymer; With the metabisulfite solution of 0.1mol/L for mobile phase; Differential refraction detector; Column temperature: 40 DEG C; Flow velocity: 0.5ml/min; Number of theoretical plate calculates should be not less than 5000 by glucose peaks.
Take the sample being about 2g containing blood clam extract, add the sherwood oil of 15 times: the solution of 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, adjust pH to 7.5 ~ 8 with sodium hydroxide solution, 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, and adds 3 times amount more than alcohol settling 6hr, abandon supernatant, subnatant is centrifugal, and precipitation adds deionized water 20ml, 80 DEG C of water-bath 2hr, centrifugal, get supernatant; Add the chloroform of 1/4 volume: the solution of normal butyl alcohol=4:1, violent jolting, centrifugal, get supernatant liquid, repetitive operation repeatedly, disappears completely to liquid level middle protein layer, gets supernatant liquid, add 3 times amount more than alcohol settling 6hr, abandon supernatant, subnatant is centrifugal, gets precipitation, less than 80 DEG C drying under reduced pressure, obtained blood clam polysaccharide.Get blood clam polysaccharide, add the flowing mutual-assistance and dissolve, make the solution of every 1ml containing 10mg, to obtain final product.
Get blood clam polysaccharide need testing solution 20 μ l, injection liquid chromatography, measure record collection of illustrative plates; If in collection of illustrative plates with the S peak in collection of illustrative plates for reference, present 2 characteristic peaks, and with S peak for 1, the relative retention time of 2 characteristic peaks is 0.56 ± 10% at peak 1, peak 2 is 0.68 ± 10%, its weight-average molecular weight (Mw) is 6.88 ~ 9.47 × 10
5between, meet blood clam polysaccharide specificity.
Meet the specificity of blood clam polypeptide and polysaccharide, explanation is blood clam simultaneously, then proceed following operation, as nothing is then judged to adulterant;
(3) blood clam determining content of peptides: take sample 0.5-2g, add 20-40 times of water, ultrasonic making is uniformly dispersed, add 10-20% trichloroacetic acid 15-40ml, mixing, leave standstill 20-50 minute, filter, get filter paper and filter residue and drying, according to " Chinese Pharmacopoeia " version in 2010 annex Ⅸ L first method, n2 method measures the content of blood clam polypeptide; We find through repetition test, and in preparation, every g blood clam extract must not be less than 0.12g containing polypeptide.
(4) blood clam determination of polysaccharide: take and be about 1-4g sample containing 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 20-50 measuring bottle, and be diluted to scale, shake up, precision measures 1-5ml, add 4% phenol solution of 1/2 volume, mixing, add rapidly sulfuric acid 5-10ml, shake up, 20-60 minute is incubated in 30-50 DEG C of water-bath, take out, put 2-10 minute in ice-water bath, take out, obtain blood clam polysaccharide test sample.Getting blood clam polysaccharide need testing solution is blank with corresponding reagent, according to UV-VIS spectrophotometry, measures absorbance at the wavelength place of 488nm.Read the weight (mg) of anhydrous dextrose in need testing solution from typical curve, calculate content.We find through repetition test, and in preparation, every g blood clam extract is containing blood clam polysaccharide in glucose (C6H12O6), must not be less than 24mg.
The sample protein matter of blood clam extract after vacuum drying and polyoses content are respectively 60% and 13%, wherein protein content is about 5 times of polyoses content, when the research of blood clam effective constituent, we find, the polypeptide of small-molecular-weight and polysaccharide are antineoplastic principal ingredient, and the protein biological activity of macromolecule is low, pharmacological effect is poor.In the research to blood clam extract, we find through test of many times, and the content of blood clam polypeptide is better effects if within the scope of 5-7 times of blood clam polyoses content.
Method of the present invention measures the content of polypeptide in blood clam, and not only specificity is strong, and provides the ratio of blood clam polypeptide and polysaccharide, can ensure that the medicine prepared with blood clam effectively and quality controllable.
For the ease of understanding the present invention, special to be further illustrated by test example below:
One, two batches of blood clam extract tablet samples assays
(1) blood clam polypeptide is differentiated:
Chromatographic condition and system flexibility: be the gel chromatographic columns of filling agent with hydrophilic spherical superpolymer; With the phosphate buffer of 0.05mol/L for mobile phase; Determined wavelength is 280 nm; Column temperature: 25 DEG C; Flow velocity 0.4ml/min; Determined wavelength 280nm; Number of theoretical plate calculates by protein molecular weight 100000 and is not less than 5000;
Take sample 10g respectively, add 12 times of water ultrasonic extraction 30 min, centrifugal 10 min, get upper liquid, filter, filtrate is mark 60%(w/v by volume) add ammonium sulfate, 0 ~ 4 DEG C of placement is spent the night, centrifugal, gets precipitation, the 10ml that adds water dissolves, and the ammonium bicarbonate soln with 1% is dislysate, is the dialysis membrane of 3500 with molecular cut off, enough hemodialysis under 0 ~ 4 DEG C of condition, is transferred to solution in dialysis membrane in 50ml measuring bottle, uses water constant volume, 0 ~ 4 DEG C of preservation, obtains blood clam polypeptide solution;
Get blood clam polypeptide solution 10 μ l respectively, injection liquid chromatography, measure, record collection of illustrative plates; 2 characteristic peaks are presented in A sample characteristic collection of illustrative plates, with the S peak in collection of illustrative plates for 1, peak 1 relative retention time is 0.58, peak 2 relative retention time is 0.69, its molecular weight is 688036,2 characteristic peaks are presented in B sample characteristic collection of illustrative plates, with the S peak in collection of illustrative plates for 1, peak 1 relative retention time is 0.55, peak 2 relative retention time is 0.63.Its molecular weight is 784503.Result shows two batches of blood clam extract tablets and all meets blood clam polysaccharide specificity, proceeds following operation.
(2) blood clam polysaccharide is differentiated:
Chromatographic condition and system flexibility: be the gel chromatographic columns of filling agent with hydrophilic spherical superpolymer; With the metabisulfite solution of 0.1mol/L for mobile phase; Differential refraction detector; Column temperature: 40 DEG C; Flow velocity: 0.5ml/min; Number of theoretical plate calculates should be not less than 5000 by glucose peaks.
Take the sample containing blood clam extract 2g respectively, add the sherwood oil of 15 times: the solution of 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, adjust pH to 7.5 ~ 8 with sodium hydroxide solution, 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, and adds 3 times amount more than alcohol settling 6hr, abandon supernatant, subnatant is centrifugal, and precipitation adds deionized water 20ml, 80 DEG C of water-bath 2hr, centrifugal, get supernatant; Add the chloroform of 1/4 volume: the solution of normal butyl alcohol=4:1, violent jolting, centrifugal, get supernatant liquid, repetitive operation repeatedly, disappears completely to liquid level middle protein layer, gets supernatant liquid, add 3 times amount more than alcohol settling 6hr, abandon supernatant, subnatant is centrifugal, gets precipitation, less than 80 DEG C drying under reduced pressure, obtained blood clam polysaccharide.Get blood clam polysaccharide, add the flowing mutual-assistance and dissolve, make the solution of every 1ml containing 10mg, to obtain final product.
Get each 20 μ l of blood clam polysaccharide need testing solution respectively, injection liquid chromatography, measure record collection of illustrative plates; Have a sharp-pointed characteristic peak at retention time 15.537min in blood clam extract formulation tablet A sample collection of illustrative plates, its molecular weight is 88070; Have a sharp-pointed characteristic peak at retention time 15.698min in blood clam extract formulation tablet B sample characteristic collection of illustrative plates, its molecular weight is 88070.Result shows the agent of two batches of blood clam extract particles and meets blood clam polypeptide specificity.
Meet the specificity of blood clam polypeptide and polysaccharide, explanation is that blood clam obtains, and proceeds assay simultaneously;
(3) blood clam determining content of peptides: take each 1.0g of sample respectively, accurately weighed, add 30 times of water, ultrasonic making is uniformly dispersed, add 12% trichloroacetic acid 30ml, mixing, leaves standstill 30 minutes, filters, get filter paper and filter residue and drying, according to " Chinese Pharmacopoeia " version in 2010 annex Ⅸ L first method, n2 method measures the content of blood clam polypeptide, calculates: every g blood clam polypeptide 0.45g in preparation A sample; Every g blood clam polypeptide 0.15g in preparation B sample.
(4) blood clam determination of polysaccharide: take containing blood clam extract 2.5g sample respectively, 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, mixing, add rapidly sulfuric acid 7ml, shake up, 30 minutes are incubated in 40 DEG C of water-baths, take out, to put in ice-water bath 5 minutes, take out, obtain blood clam polysaccharide test sample.Getting blood clam polysaccharide need testing solution is blank with corresponding reagent, according to UV-VIS spectrophotometry, measures absorbance at the wavelength place of 488nm.Read the weight (mg) of anhydrous dextrose in need testing solution from typical curve, calculate: 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.
In preparation A sample, blood clam content of peptides is 5 times that blood clam polysaccharide contains, and in preparation B sample, blood clam content of peptides is 10 times that blood clam polysaccharide contains.
Two, two batches of blood clam extract tablet drug effects compare
Test method: choose healthy mice 10, inoculation lung cancer cell line (2*105/ only), after inoculation 14d, peels off tumour under aseptic condition, makes cell suspension with Potter-Elvehjem Tissue Grinders.By healthy mice 30, be divided into 3 groups at random, the equal tail vein of each group containing cell number 1*105/ only accepts the cell suspension 0.2ml(of above-mentioned preparation).Give preparation A, B 0.25g/kg intravenous injection respectively, blank group injection normal saline.Each group all once a day, continuous 10 days.
Observation item: stop observation post administration and respectively organize body weight mass change, survival day, calculates survival prolongation rate LS=[(medication group survival day/control group survival day)]-1*100% as follows.Put to death animal and claim lung weight, calculate the heavy index of lung.The ratio * 100% of heavy (the mg)/body weight (10g) of the heavy index=mouse lung of lung.
The results are shown in Table:
Preparation A, B are all better than blank group in survival day, survival prolongation rate, the heavy index of lung, and preparation A curative effect is better than preparation B, shows blood clam polypeptide and polyoses content better efficacy in certain proportion in blood clam extract tablet.
Below in conjunction with embodiment, the invention will be further described.
Embodiment 1
Get fresh blood clam meat, broken, cross 40 mesh sieves, add 1 times of water gaging, below 10 DEG C, stir extraction 4 hours, extracting liquid filtering, centrifugal.Get above-mentioned centrifugate freeze-drying, obtain blood clam extract.
A sharp-pointed characteristic peak " peak S " is had at retention time 15.564min in blood clam extract characteristic spectrum.Its molecular weight is 88070.Meet blood clam polypeptide specificity, proceed following operation;
Present 2 characteristic peaks in blood clam extract characteristic spectrum, with the S peak in collection of illustrative plates for 1, peak 1 relative retention time is 0.57, peak 2 relative retention time is 0.68, its molecular weight is 696214, meets blood clam polysaccharide specificity.
Meet the specificity of blood clam polypeptide and polysaccharide, explanation is that blood clam obtains, and proceeds assay simultaneously;
Get blood clam extract porphyrize, precision takes 2.012g, and add 30 times of water, ultrasonic making is uniformly dispersed, and adds 10% trichloroacetic acid 45ml, mixing, leaves standstill 45 minutes, filters, gets filter paper and filter residue and drying, obtain test sample.Measured according to n2 method (" Chinese Pharmacopoeia " version in 2010 annex Ⅸ L first method) by test sample, the every g of blood clam extract is containing blood clam polypeptide 0.46g.
Take containing blood clam extract 2.601g, put in round-bottomed flask, add 42 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 10 minutes, precipitation is dissolved in water, and puts in 50ml measuring bottle, and is diluted to scale, shake up, precision measures 2ml, adds 4% phenol solution of 1/2 volume, mixing, add rapidly sulfuric acid 8ml, shake up, in 40 DEG C of water-baths, be incubated 30 minutes, take out, to put in ice-water bath 5 minutes, take out, obtain blood clam polysaccharide test sample.Getting blood clam polysaccharide need testing solution is blank with corresponding reagent, according to UV-VIS spectrophotometry, measures absorbance at the wavelength place of 488nm.Read the weight (mg) of anhydrous dextrose in need testing solution from typical curve, calculate the every g of blood clam extract containing polysaccharide 72mg.
In preparation, blood clam content of peptides is 6.39 times that blood clam polysaccharide contains.
Embodiment 2
Get dry blood clam meat, pulverize, cross 80 mesh sieves, add the water of 10 times amount, heating extraction 2 times, each 1 hour, merge extract, filter, get filtrate, being concentrated into relative density is 1.04 ~ 1.06(60 DEG C) time, centrifugal (2000 revs/min).Get above-mentioned centrifugate less than 80 DEG C, drying under reduced pressure, pulverize, obtain blood clam extract.Extract is prepared into tablet.
Get tablet and carry out the discriminating of blood clam polypeptide, in characteristic spectrum, have a sharp-pointed characteristic peak " peak S " at retention time 15.239min.Its molecular weight is 88070.Meet blood clam polypeptide specificity, proceed following operation;
Get tablet and carry out the discriminating of blood clam polysaccharide, present 2 characteristic peaks in characteristic spectrum, with the S peak in collection of illustrative plates for 1, peak 1 relative retention time is 0.59, peak 2 relative retention time is 0.73, its molecular weight is 725444, meets blood clam polysaccharide specificity.
Meet the specificity of blood clam polypeptide and polysaccharide, explanation is that blood clam obtains, and proceeds assay simultaneously;
Get tablet porphyrize prepared by blood clam extract, precision takes 1.512g, and containing blood clam extract 1.218g, add 21 times of water, ultrasonic making is uniformly dispersed, and adds 19% trichloroacetic acid 30ml, mixing, leaves standstill 33 minutes, filters, gets filter paper and filter residue and drying, obtain test sample.Measured according to n2 method (" Chinese Pharmacopoeia " version in 2010 annex Ⅸ L first method) by test sample, the every g of blood clam extract is containing blood clam polypeptide 0.23g.
Get tablet porphyrize prepared by blood clam extract, precision takes 3.532g, containing blood clam extract 2.826g, put in round-bottomed flask, add 39 times of water, add hot reflux 1 hour, filter with absorbent cotton, filtrate moves in 100ml measuring bottle, add water to scale, shake up, precision measures 1ml, add 6 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 2ml, add 4% phenol solution of 1/2 volume, mixing, add rapidly sulfuric acid 5ml, shake up, 39 minutes are incubated in 40 DEG C of water-baths, take out, to put in ice-water bath 50 minutes, take out, obtain blood clam polysaccharide test sample.Getting blood clam polysaccharide need testing solution is blank with corresponding reagent, according to UV-VIS spectrophotometry, measures absorbance at the wavelength place of 488nm.Read the weight (mg) of anhydrous dextrose in need testing solution from typical curve, calculate the every g of blood clam extract containing blood clam polysaccharide 42mg.
In preparation, blood clam content of peptides is 5.18 times that blood clam polysaccharide contains.
Embodiment 3
Prepare blood clam extract according to the blood clam method for preparing extractive in embodiment 2, extract is made capsule.
Get capsule 's content and carry out the discriminating of blood clam polypeptide, have a sharp-pointed characteristic peak " peak S " at retention time 15.933min in result display blood clam extract characteristic spectrum, its molecular weight is 88070.Meet blood clam polypeptide specificity, proceed following operation;
Get capsule 's content and carry out the discriminating of blood clam polypeptide, present 2 characteristic peaks in characteristic spectrum, with the S peak in collection of illustrative plates for 1, peak 1 relative retention time is 0.53, peak 2 relative retention time is 0.63, its molecular weight is 786213, meets blood clam polysaccharide specificity.
Meet the specificity of blood clam polypeptide and polysaccharide, explanation is that blood clam obtains, and proceeds assay simultaneously;
Get capsule 's content and carry out the every g of assay blood clam extract containing blood clam polypeptide 0.35g.Every g is containing blood clam polysaccharide 65mg.
In preparation, blood clam content of peptides is 5.38 times that blood clam polysaccharide contains.
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 and carry out the discriminating of blood clam polypeptide, concrete steps are as follows:
Chromatographic condition and system flexibility: be the gel chromatographic columns of filling agent with hydrophilic spherical superpolymer; With the phosphate buffer of 0.05 mol/L for mobile phase; Determined wavelength is 280 nm; Column temperature: 25 DEG C; Flow velocity: 0.4ml/min.Number of theoretical plate is calculated as 8000 by protein molecular weight 100000.
Capsule 's content porphyrize is got in the preparation of blood clam extract solution, take 10g, add 12 times of water ultrasonic extraction 30 min, centrifugal 10 min, get upper liquid, filter, filtrate is mark 60%(w/v by volume) add ammonium sulfate, 0 ~ 4 DEG C of placement is spent the night, centrifugal, get precipitation, the 10ml that adds water dissolves, ammonium bicarbonate soln with 1% is dislysate, be the dialysis membrane of 3500 with molecular cut off, enough hemodialysis (the BaCl2 solution with 1% checks whether sulfate ion dialyses completely) under 0 ~ 4 DEG C of condition, solution in dialysis membrane is transferred in 50ml measuring bottle, use water constant volume, 0 ~ 4 DEG C of preservation, obtain blood clam blood clam polypeptide test sample.
Get blood clam protein (polypeptide) test sample solution 20 μ l, injection liquid chromatography, measure.
A sharp-pointed characteristic peak " peak S " is had at retention time 15.623min in characteristic spectrum.Its molecular weight is 88070, meets blood clam polypeptide specificity, proceeds following operation;
Get granule and carry out the discriminating of blood clam polysaccharide, concrete steps are as follows:
Chromatographic condition and system flexibility: be the gel chromatographic columns of filling agent with hydrophilic spherical superpolymer; With the metabisulfite solution of 0.1mol/L for mobile phase; Differential refraction detector; Column temperature: 40 DEG C; Flow velocity: 0.5ml/min; Number of theoretical plate calculates should be not less than 5000 by glucose peaks.
Take containing blood clam extract particles 2g, add the sherwood oil of 15 times respectively: the solution of 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, adjust pH to 7.5 ~ 8 with sodium hydroxide solution, 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, and adds 3 times amount more than alcohol settling 6hr, abandon supernatant, subnatant is centrifugal, and precipitation adds deionized water 20ml, 80 DEG C of water-bath 2hr, centrifugal, get supernatant; Add the chloroform of 1/4 volume: the solution of normal butyl alcohol=4:1, violent jolting, centrifugal, get supernatant liquid, repetitive operation repeatedly, disappears completely to liquid level middle protein layer, gets supernatant liquid, add 3 times amount more than alcohol settling 6hr, abandon supernatant, subnatant is centrifugal, gets precipitation, less than 80 DEG C drying under reduced pressure, obtained blood clam polysaccharide.Get blood clam polysaccharide, add the flowing mutual-assistance and dissolve, make the solution of every 1ml containing 10mg, to obtain final product.
Get each 20 μ l of blood clam polysaccharide need testing solution, injection liquid chromatography, measure record collection of illustrative plates; Present 2 characteristic peaks in collection of illustrative plates, with the S peak in collection of illustrative plates for 1, peak 1 relative retention time is 0.61, peak 2 relative retention time is 0.72, its molecular weight is 844021.Meet blood clam polysaccharide specificity.
Meet the specificity of blood clam polypeptide and polysaccharide, explanation is that blood clam obtains, and proceeds assay simultaneously.
Get granule and carry out the every g of assay blood clam extract containing blood clam polypeptide 0.63g.Every g is containing blood clam polysaccharide 102mg.
In preparation, blood clam content of peptides is 6.18 times that blood clam polysaccharide contains.
Embodiment 5
Get the blood clam of Different sources respectively, prepare 3 batches of blood clam extract pills by the method for embodiment 2, extract sample A, sample B and the sample C of three crowdes respectively, carry out differentiating and assay.3 batches of measurement results are:
Have a sharp-pointed characteristic peak " peak S " at retention time 15.652min in sample A blood clam polypeptide diagnostic characteristics collection of illustrative plates, its molecular weight is 88070, meets blood clam polypeptide specificity.Present 2 characteristic peaks in blood clam polypeptide diagnostic characteristics collection of illustrative plates, with the S peak in collection of illustrative plates for 1, peak 1 relative retention time is 0.54, peak 2 relative retention time is 0.66, its molecular weight is 798745, meets blood clam polysaccharide specificity.Meet the specificity of blood clam polypeptide and polysaccharide, explanation is that blood clam obtains, and proceeds assay simultaneously.The every g of assay blood clam extract is containing blood clam polypeptide 0.66g.Every g is containing blood clam polysaccharide 99mg.In preparation, blood clam content of peptides is 6.67 times that blood clam polysaccharide contains.
Have a sharp-pointed characteristic peak " peak S " at retention time 15.231min in sample B blood clam polypeptide diagnostic characteristics collection of illustrative plates, its molecular weight is 88070, meets blood clam polypeptide specificity.Present 2 characteristic peaks in blood clam polypeptide diagnostic characteristics collection of illustrative plates, with the S peak in collection of illustrative plates for 1, peak 1 relative retention time is 0.56, peak 2 relative retention time is 0.69, its molecular weight is 801214, meets blood clam polysaccharide specificity.Meet the specificity of blood clam polypeptide and polysaccharide, explanation is that blood clam obtains, and proceeds assay simultaneously.Get granule and carry out the every g of assay blood clam extract containing blood clam polypeptide 0.53g.Every g is containing blood clam polysaccharide 102mg.In preparation, blood clam content of peptides is 5.20 times that blood clam polysaccharide contains.
Have a sharp-pointed characteristic peak " peak S " at retention time 15.762min in sample C blood clam polypeptide diagnostic characteristics collection of illustrative plates, its molecular weight is 88070, meets blood clam polypeptide specificity.Present 2 characteristic peaks in blood clam polypeptide diagnostic characteristics collection of illustrative plates, with the S peak in collection of illustrative plates for 1, peak 1 relative retention time is 0.53, peak 2 relative retention time is 0.65, its molecular weight is 754102, meets blood clam polysaccharide specificity.Meet the specificity of blood clam polypeptide and polysaccharide, explanation is that blood clam obtains, and proceeds assay simultaneously.Get granule and carry out the every g of assay blood clam extract containing blood clam polypeptide 0.32g.Every g is containing blood clam polysaccharide 59mg.In preparation, blood clam content of peptides is 5.42 times that blood clam polysaccharide contains.
Claims (6)
1. the content assaying method of a blood clam, first by the polypeptide in gel chromatographic columns high performance liquid chromatography discriminating sample and polysaccharide, if two specificities are all satisfied, then determine it is blood clam, measure the content of polypeptide and polysaccharide in blood clam again, it is characterized in that: wherein the discrimination method of blood clam polypeptide uses gel chromatographic columns high performance liquid chromatography, and concrete steps are as follows:
Chromatographic condition and system flexibility: be the gel chromatographic columns of filling agent with hydrophilic spherical superpolymer; With the phosphate buffer of 0.05mol/L for mobile phase; Determined wavelength is 280 nm; Column temperature: 25 DEG C; Flow velocity 0.4ml/min; Determined wavelength 280nm; Number of theoretical plate calculates by protein molecular weight 100000 and is not less than 5000;
Take sample 10g, add 12 times of water ultrasonic extraction 30 min, centrifugal 10 min, get upper liquid, filter, filtrate is mark 60%, w/v by volume, add ammonium sulfate, 0 ~ 4 DEG C of placement is spent the night, centrifugal, get precipitation, the 10ml that adds water dissolves, and the ammonium bicarbonate soln with 1% is dislysate, be the dialysis membrane of 3500 with molecular cut off, enough hemodialysis under 0 ~ 4 DEG C of condition, is transferred to solution in dialysis membrane in 50ml measuring bottle, use water constant volume, 0 ~ 4 DEG C of preservation, obtains blood clam polypeptide solution;
Get blood clam polypeptide solution 10 μ l, injection liquid chromatography, measure, record collection of illustrative plates; If there is the characteristic peak of blood clam polypeptide in collection of illustrative plates within retention time 15.6min ± 5%, its molecular weight is 88070, meets blood clam polypeptide specificity, as nothing is then judged to adulterant.
2. the content assaying method of blood clam according to claim 1, it is characterized in that the discrimination method of wherein blood clam polysaccharide uses gel chromatographic columns high performance liquid chromatography, concrete steps are as follows:
Chromatographic condition and system flexibility: be the gel chromatographic columns of filling agent with hydrophilic spherical superpolymer; With the metabisulfite solution of 0.1mol/L for mobile phase; Differential refraction detector; Column temperature: 40 DEG C; Flow velocity: 0.5ml/min; Number of theoretical plate calculates should be not less than 5000 by glucose peaks;
Take the sample being about 2g containing blood clam extract, add the sherwood oil of 15 times: the solution of 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, adjust pH to 7.5 ~ 8 with sodium hydroxide solution, 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, and adds 3 times amount more than alcohol settling 6hr, abandon supernatant, subnatant is centrifugal, and precipitation adds deionized water 20ml, 80 DEG C of water-bath 2hr, centrifugal, get supernatant; Add the chloroform of 1/4 volume: the solution of normal butyl alcohol=4:1, violent jolting, centrifugal, get supernatant liquid, repetitive operation repeatedly, disappears completely to liquid level middle protein layer, gets supernatant liquid, add 3 times amount more than alcohol settling 6hr, abandon supernatant, subnatant is centrifugal, gets precipitation, less than 80 DEG C drying under reduced pressure, obtained blood clam polysaccharide; Get blood clam polysaccharide, add the flowing mutual-assistance and dissolve, make the solution of every 1ml containing 10mg, to obtain final product;
Get blood clam polysaccharide need testing solution 20 μ l, injection liquid chromatography, measure record collection of illustrative plates; If in collection of illustrative plates with the S peak in collection of illustrative plates for reference, present 2 characteristic peaks, and with S peak for 1, the relative retention time of 2 characteristic peaks is 0.56 ± 10% at peak 1, peak 2 is 0.68 ± 10%, its weight-average molecular weight Mw is 6.88 × 10
5~ 9.47 × 10
5between, meet blood clam polysaccharide specificity;
Meet the specificity of blood clam polypeptide and polysaccharide, explanation is blood clam simultaneously, as nothing is then judged to adulterant.
3. the content assaying method of blood clam according to claim 1, it is characterized in that the content assaying method of wherein blood clam polysaccharide uses nitriding, concrete steps are as follows:
Take sample 0.5-2g, add 20-40 times of water, ultrasonic making is uniformly dispersed, and adds 10-20% trichloroacetic acid 15-40ml, mixing, leaves standstill 20-50 minute, filters, get filter paper and filter residue and drying, according to " Chinese Pharmacopoeia " version in 2010 annex Ⅸ L first method, n2 method measures the content of blood clam polypeptide; In preparation, every g blood clam extract must not be less than 0.12g containing polypeptide.
4. the content assaying method of blood clam according to claim 1, it is characterized in that the content assaying method of wherein blood clam polypeptide uses spectrophotometric method, concrete steps are as follows:
Take containing blood clam extract 1-4g sample, 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 20-50 measuring bottle, and be diluted to scale, shake up, precision measures 1-5ml, add 4% phenol solution of 1/2 volume, mixing, add rapidly sulfuric acid 5-10ml, shake up, 20-60 minute is incubated in 30-50 DEG C of water-bath, take out, put 2-10 minute in ice-water bath, take out, obtain blood clam polysaccharide test sample, getting blood clam polysaccharide need testing solution is blank with corresponding reagent, according to UV-VIS spectrophotometry, measures 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, every g blood clam extract is containing blood clam polysaccharide in glucose C6H12O6, must not be less than 24mg.
5. according to the content assaying method of claim 1 or claim 3 or blood clam according to claim 4, it is characterized in that the content of blood clam polypeptide be the 5-7 of blood clam polyoses content doubly.
6., according to the content assaying method of claim 1 or claim 3 or blood clam according to claim 4, it is characterized in that the assay for the various preparation of blood clam extract.
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Non-Patent Citations (5)
| Title |
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| A novel yeast-binding lectin from hemolymph Cyclina sinensis (Gmelin) and its effects on yeast cells;Changqing Tong等;《Process Biochemistry》;20120817;第47卷;2166–2171 * |
| Determination of L-Ephedrine, Pseudoephedrine,and Caffeine in Rat Plasma by Liquid Chromatography–Tandem Mass Spectrometry;Stephen D. Cooper等;《Journal of Analytical Toxicology》;20110831;第35卷;341-348 * |
| 毛蚶天然多肽提取纯化及其体外抗氧化能力和抑制肿瘤活性的初步研究;刘思聪;《中国优秀硕士学位论文全文数据库(工程科技Ⅰ辑)》;20140315;第2.2.4节 * |
| 海洋生物中牛磺酸、多糖、肽等活性物质的提取研究进展;钱清华;《食品工业科技》;20131231;第34卷(第13期);383-387 * |
| 邓一清.翡翠贻贝多糖的分离纯化、成分测定和保湿性研究.《中国优秀硕士学位论文全文数据库(农业科技辑)》.2013,论文第8-9页"2.3 方法". * |
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