CN103954704A - Method for assaying content of blood clam protein - Google Patents
Method for assaying content of blood clam protein Download PDFInfo
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- CN103954704A CN103954704A CN201410186505.1A CN201410186505A CN103954704A CN 103954704 A CN103954704 A CN 103954704A CN 201410186505 A CN201410186505 A CN 201410186505A CN 103954704 A CN103954704 A CN 103954704A
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Abstract
The invention relates to a method for assaying the content of blood clam protein. The method is characterized in that firstly, polypeptide in an assayed sample is determined as the blood clam protein through a high efficiency liquid chromatographic method by use of a gel chromatographic column and then the content is assayed through a nitrogen determination method. In a preparation, every 1g of blood clam extract contains not less than 0.12g of blood clam polypeptide, and the higher the content is, the better the curative effect is. The method can be used for assaying the content of various preparations of the blood clam extract to guarantee the product quality of the blood clam preparations during production and use, and thus the curative effect of medicaments is guaranteed.
Description
Technical field
The present invention relates to medicine detection method, be specifically related to a kind of content assaying method of blood clam protein, belong to field of pharmaceutical technology.
Background technology
Blood clam meat is containing rich in protein, vitamin and the necessary amino acid of 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 shows that blood clam has antitumor action, has effect of removing interior free yl, improving 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.
One of main bioactive ingredients of blood clam according to bibliographical information blood clam polypeptide, have protect the liver, antitumor, regulate immune effect.The doctoral advisor Yu Rongmin of Ji'nan University professor report on January 5th, 2011, fresh blood clam shells, after homogenate, centrifugal, dialysis, freeze-drying, make blood clam polypeptide extract, method is measured the impact on 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 variation in mouse spleen lymphocyte cycle; On the impact of major cytokine IFN-γ and IL-4 secretion level.Result blood clam polypeptide extract can significantly promote mice spleen lymphocytes proliferation, strengthens Turnover of Mouse Peritoneal Macrophages and engulfs dimethyl diaminophenazine chloride activity and mouse NK cell killing activity; Promote splenic lymphocyte G0/G1 phase to DNA synthesis phase (s phase) to transform; Collaborative ConA effect, can increase the secretion of IFN-γ, and can suppress the secretion of IL-4.The expensive report that waits of the Dou Chang of China Medicine University; with blood clam meat hydrolyzate mouse stomach; result: the active rising of serum glutamic pyruvic transminase that phenixin, thio-ethylamine and prednisolone are caused all has obvious reducing effect, shows that blood clam hydrolyzate has significant protective effect to Experimental Hepatic Damage.
We test and find that blood clam polypeptide can obviously suppress the expression of hepatitis B surface antigen and E antigen, and hepatitis B DNA is had to inhibiting effect.
Protein is the base substance of vital movement, all animals and plants life entities all contain proteins and peptides, in the situation that cannot determining protein source, just can not ensure the validity of medicine, content that can not Accurate Determining protein, 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 polypeptide 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 polypeptide is provided, make blood clam preparation in producing and using, effectively ensure product quality.
A content assaying method for blood clam protein and peptide, is characterized in that first determining that by gel chromatographic columns high performance liquid chromatography the polypeptide in sample is blood clam polypeptide, then measures content with nitriding, 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 phosphate buffer of 0.05mol/L as mobile phase; Detecting wavelength is 280 nm; Column temperature: 25 DEG C; Flow velocity 0.4ml/min; Detect wavelength 280nm; Number of theoretical plate calculates and is not less than 5000 by protein molecular weight 100000;
(2) take sample 10g, add 12 times of ultrasonic extraction 30 min of water, centrifugal 10 min, get upper strata 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 taking 1% is dislysate, the dialysis membrane that is 3500 with molecular cut off, under 0～4 DEG C of condition, fully dialysis, is transferred to solution in dialysis membrane in 50ml measuring bottle water constant volume, 0～4 DEG C of preservation, obtains blood clam polypeptide solution;
(3) get blood clam polypeptide solution 10 μ l, injection liquid chromatography, measures, and records 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, proceeds following operation, as nothing is judged to adulterant;
(4) take sample 0.5-2g, add 20-40 times of water, ultrasonic making is uniformly dispersed, add 10-20% trichloroacetic acid 15-40ml, mix, leave standstill 20-50 minute, filter, get filter paper and filter residue and drying, according to annex IX L first method of " Chinese Pharmacopoeia " version in 2010, n2 method is measured the content of blood clam polypeptide;
We are through repetition test discovery, and in preparation, every g blood clam extract must not be less than 0.12g containing polypeptide, and content is higher, better efficacy.
The sample protein matter content of blood clam extract after vacuum drying is 60%, and in the time of the research of blood clam effective constituent, we find, the polypeptide of little molecular weight is antineoplastic principal ingredient, and the protein biological activity of macromolecule is low, and pharmacological effect is poor.And the blood clam protein of macromolecule and stalwart blood clam, mud blood clam, flower clam, Scallop Extract, its contained polypeptide molecular weight is different from blood clam polypeptide, use gel chromatographic columns high effective liquid chromatography for measuring, in collection of illustrative plates within retention time 15.6min ± 5% characteristic peak without blood clam polypeptide, therefore strong by the content specificity of method mensuration blood clam polypeptide 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 extract tablet samples assays
(1) chromatographic condition and system flexibility: the gel chromatographic columns that is filling agent with hydrophilic spherical superpolymer; Taking the phosphate buffer of 0.05mol/L as mobile phase; Detecting wavelength is 280 nm; Column temperature: 25 DEG C; Flow velocity 0.4ml/min; Detect wavelength 280nm; Number of theoretical plate calculates and is not less than 5000 by protein molecular weight 100000;
(2) take the each 10g of sample, add respectively 12 times of ultrasonic extraction 30 min of water, centrifugal 10 min, get upper strata 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 taking 1% is dislysate, the dialysis membrane that is 3500 with molecular cut off, under 0～4 DEG C of condition, fully dialysis, is transferred to solution in dialysis membrane in 50ml measuring bottle water constant volume, 0～4 DEG C of preservation, obtains blood clam polypeptide solution;
(3) get blood clam polypeptide solution 10 μ l, injection liquid chromatography, measures, and records collection of illustrative plates; In blood clam extract formulation tablet A sample collection of illustrative plates, have a sharp-pointed characteristic peak at retention time 15.537min, its molecular weight is 88070; In blood clam extract formulation tablet B sample characteristic collection of illustrative plates, have a sharp-pointed characteristic peak at retention time 15.698min, its molecular weight is 88070.Result shows that two batches of blood clam extract tablets are all to make with blood clam.Proceed assay;
(4) take the each 0.5-2g of sample, add respectively 20-40 times of water, ultrasonic making is uniformly dispersed, and adds 10-20% trichloroacetic acid 15-40ml, mixes, leave standstill 20-50 minute, filter, get filter paper and filter residue and drying, according to annex IX L first method of " Chinese Pharmacopoeia " version in 2010, n2 method is measured the content of blood clam polypeptide, calculates: every g blood clam polypeptide 0.45g in preparation A sample; Every g blood clam polypeptide 0.15 in preparation B sample.
Two, two batches of blood clam extract tablet drug effects comparison
Test method: choose 10 of healthy mices, 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 30 of healthy mices, be divided at random 3 groups, containing cell number 1*105/ only respectively organize cell suspension 0.2ml(that equal tail vein accepts above-mentioned preparation).Give respectively preparation A, B 0.25g/kg intravenous injection, blank group injection equivalent physiological 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 existence rate elongation LS=[(medication group survival day/control group survival day as follows)]-1*100%.Put to death animal and claim lung weight, calculate the heavy index of lung.The ratio * 100% of heavy heavy (the mg)/body weight of index=mouse lung of lung (10g).
The results are shown in Table:
Preparation A, B are all being better than blank group aspect survival day, the heavy index of existence rate elongation, lung, and the high preparation A curative effect of content is better than preparation B, show that blood clam extract tablet anticancer effect is definite, and dosage are higher, and effect is better.
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, extracts 4 hours extracting liquid filtering, centrifugal (2000 revs/min) 10 DEG C of following stirrings.Get above-mentioned centrifugate freeze-drying, obtain blood clam extract.
In blood clam extract characteristic spectrum, there is a sharp-pointed characteristic peak " peak S " at retention time 15.537min.Its molecular weight is 88070.Result shows that blood clam extract is to make with blood clam.Proceed assay.
Get blood clam extract porphyrize, precision takes 1.985g, adds 30 times of water, and ultrasonic making is uniformly dispersed, and adds 10% trichloroacetic acid 40ml, mixes, and leaves standstill 50 minutes, filters, and gets filter paper and filter residue and drying, obtains test sample.Test sample is measured according to n2 method (annex IX L first method of " Chinese Pharmacopoeia " version in 2010), and the every g of blood clam extract is containing blood clam polypeptide 0.25g.
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 (2000 revs/min).Get above-mentioned centrifugate below 80 DEG C, drying under reduced pressure, pulverizes, and obtains blood clam extract.Extract is made to tablet.
In blood clam extract tablet characteristic spectrum, there is a sharp-pointed characteristic peak " peak S " at retention time 15.537min.Its molecular weight is 88070.Result shows that blood clam extract tablet is to make with blood clam, proceeds assay.
Get tablet porphyrize prepared by blood clam extract, precision takes 1.523g, containing blood clam extract 1.218g, adds 20 times of water, and ultrasonic making is uniformly dispersed, and adds 20% trichloroacetic acid 30ml, mixes, and leaves standstill 35 minutes, filters, and gets filter paper and filter residue and drying, obtains test sample.Test sample is measured according to n2 method (annex IX L first method of " Chinese Pharmacopoeia " version in 2010), and the every g of blood clam extract is containing blood clam polypeptide 0.16g.
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, have a sharp-pointed characteristic peak " peak S " at retention time 15.537min.Its molecular weight is 88070, and result shows that blood clam extract tablet is to make with blood clam, proceeds assay.
Get capsule 's content porphyrize, precision takes 0.572, containing blood clam extract 0.515g, adds 40 times of water, and ultrasonic making is uniformly dispersed, and adds 15% trichloroacetic acid 15ml, mixes, and leaves standstill 20 minutes, filters, and gets filter paper and filter residue and drying, obtains test sample.Test sample is measured according to n2 method (annex IX L first method of " Chinese Pharmacopoeia " version in 2010), and the every g of blood clam extract is containing blood clam polypeptide 0.06g.
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 phosphate buffer of 0.05 mol/L as mobile phase; Detecting 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 ultrasonic extraction 30 min of water, centrifugal 10 min, get upper strata 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 taking 1% is dislysate, the dialysis membrane that is 3500 with molecular cut off, fully dialysis under 0～4 DEG C of condition (the BaCl2 solution with 1% checks whether sulfate ion dialyses completely), solution in dialysis membrane is transferred in 50ml measuring bottle, water constant volume, 0～4 DEG C of preservation, obtain blood clam polypeptide test sample.
Get blood clam protein (polypeptide) test sample solution 20 μ l, injection liquid chromatography, measures.
In characteristic spectrum, there is a sharp-pointed characteristic peak " peak S " at retention time 15.537min.Its molecular weight is 88070, and result shows that the agent of blood clam extract particles is to make with blood clam, proceeds assay.
Get granule porphyrize, precision takes 2.139g, containing blood clam extract 1.925g, adds 19.25ml water, and ultrasonic making is uniformly dispersed, and adds 13% trichloroacetic acid 33ml, mixes, and leaves standstill 40 minutes, filters, and gets filter paper and filter residue and drying, obtains test sample.Test sample is measured according to n2 method (annex IX L first method of " Chinese Pharmacopoeia " version in 2010), and the every g of blood clam extract is containing blood clam polypeptide 0.137g.
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, have a sharp-pointed characteristic peak " peak S " at retention time 15.347min, its molecular weight is 88070, and result shows that pill is to make with blood clam.Proceed assay.The every g of blood clam extract is containing blood clam polypeptide 1.986g.
In sample B characteristic spectrum, have a sharp-pointed characteristic peak " peak S " at retention time 15.854min, its molecular weight is 88070, and result shows that pill is to make with blood clam.Proceed assay.The every g of blood clam extract is containing blood clam polypeptide 1.681g.
In sample A characteristic spectrum, have a sharp-pointed characteristic peak " peak S " at retention time 15.622min, its molecular weight is 88070, and result shows that pill is to make with blood clam.Proceed assay.The every g of blood clam extract is containing blood clam polypeptide 0.678g.
Claims (5)
1. a content assaying method for blood clam protein and peptide, is characterized in that first determining that by gel chromatographic columns high performance liquid chromatography the polypeptide in sample is blood clam polypeptide, then measures content with nitriding.
2. determine that according to the gel chromatographic columns high performance liquid chromatography of claim 1 polypeptide in sample is blood clam polypeptide, 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 phosphate buffer of 0.05mol/L as mobile phase; Detecting wavelength is 280 nm; Column temperature: 25 DEG C; Flow velocity 0.4ml/min; Detect wavelength 280nm; Number of theoretical plate calculates and is not less than 5000 by protein molecular weight 100000;
(2) take sample 10g, add 12 times of ultrasonic extraction 30 min of water, centrifugal 10 min, get upper strata 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 taking 1% is dislysate, the dialysis membrane that is 3500 with molecular cut off, under 0～4 DEG C of condition, fully dialysis, is transferred to solution in dialysis membrane in 50ml measuring bottle water constant volume, 0～4 DEG C of preservation, obtains blood clam polypeptide solution;
(3) get blood clam polypeptide solution 10 μ l, injection liquid chromatography, measures, and records collection of illustrative plates; If there is blood clam polypeptide in collection of illustrative plates within retention time 15.6min ± 5%.
3. measure content method according to the nitriding of claim 1, it is characterized in that taking sample 0.5-2g, add 20-40 times of water, ultrasonic making is uniformly dispersed, and adds 10-20% trichloroacetic acid 15-40ml, mix, leave standstill 20-50 minute, filter, get filter paper and filter residue and drying, according to annex IX L first method of " Chinese Pharmacopoeia " version in 2010, n2 method is measured the content of blood clam polypeptide.
4. according to the content assaying method of a kind of blood clam protein and peptide of claim 1 or claim 3, it is characterized in that in preparation, every g blood clam extract must not be less than 0.12g containing blood clam polypeptide, content is higher, better efficacy.
5. according to the content assaying method of a kind of blood clam protein and peptide 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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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104825496A (en) * | 2015-04-19 | 2015-08-12 | 青岛大学 | Blood clam extract and application thereof in medicaments for promoting postpartum uterine contraction |
| CN110632229A (en) * | 2019-09-24 | 2019-12-31 | 华兰生物工程重庆有限公司 | Method for detecting protein content of human immunoglobulin (pH4) infused in static environment by high performance liquid chromatograph |
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Address after: 250014 81 thousand Buddha Shandong Road, Lixia District, Ji'nan, Shandong Patentee after: Shandong Zhonghong Kang Pharmaceutical Technology Development Co Ltd Address before: 250014 No. 4, West building, Shandong Academy of Sciences, 19 Shandong Road, Lixia Road, Lixia District, Shandong Patentee before: Jinan Kangzhong Medicin Science & Technology Co., Ltd. |