CN102253036A - Filtration interception method for detecting trace amounts of proteins in traditional Chinese medicine injection - Google Patents
Filtration interception method for detecting trace amounts of proteins in traditional Chinese medicine injection Download PDFInfo
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Abstract
The invention discloses a filtration interception method for detecting trace of proteins in a traditional Chinese medicine injection. The method can control a quality of a traditional Chinese medicine injection through detecting trace amounts of protein impurities. The method is characterized by comprising the following steps that 1) a protein adsorption film is installed on a filter; 2) a traditional Chinese medicine injection passes through the protein adsorption film; 3) the protein adsorption film treated through the step 2 is washed by an organic solvent-containing solution, and 4) the washed protein adsorption film is treated through coomassie brilliant blue staining. When a protein content of a traditional Chinese medicine injection is less than 15 ng/mL, the method can still carry out protein detecting, and has a strong capacity of resisting disturbance, thus is suitable for detecting trace amounts of proteins in a traditional Chinese medicine injection.
Description
Affiliated technical field
The present invention relates to traditional Chinese medicine impurity detection range, be used for the protein-based impurity of traditional Chinese medicine, with the quality of control traditional Chinese medicine.
Technical background
Traditional Chinese medicine is created in the forties in last century the earliest, is started by scholars such as Qian Xinzhong.In China, traditional Chinese medicine has been widely used in clinical and has obtained the approval of industry.One one of the version Pharmacopoeia of the People's Republic of China in 2010 (be called for short " Chinese pharmacopoeia) has just been included folk prescription and compound injections such as erigeron breviscapus, QINGKAILING, 'Zhichuanling.Traditional Chinese medicine is advancing the form of Chinese drug development, expands the range of application of Chinese medicine, performs meritorious deeds never to be obliterated really in aspects such as service numerous people health.Safe, effective, quality controllable still is the gross requirement of traditional Chinese medicine.Technologically speaking, industry has solved the validity problem (although mechanism of action is still not exclusively removed) of traditional Chinese medicine substantially, and safety issue still is an industry difficult problem.
Yet, along with traditional Chinese medicine be widely used in clinically, its bad reaction exposed day by day, particularly lethal bad reaction also have report repeatly.According to present disclosed bad reaction data, the bad reaction of traditional Chinese medicine can be involved each system and internal organs, even causes death.The bad reaction of a lot of traditional Chinese medicines and original pharmacological action are irrelevant, and according to multianalysis, these bad reactions mainly belong to hypersensitivity, i.e. pathologic immune response comprises I, II, III and the immune response of IV type pathologic.The pathologic immune response is still immune response, and the participation of antigen must be arranged, and its antigen can be haptens or comlete antigen.Haptens belongs to the micromolecule composition more, and comlete antigen mostly is macromolecular components, see so that protein more, and the antigenicity of protein type is the strongest, and the hypersensitivity of initiation is comparatively serious.Therefore the antigenicity composition that reduces in the traditional Chinese medicine is one of Key Strategy that improves the traditional Chinese medicine security.
Traditional Chinese medicine mostly is plant extracts greatly.As bio-extract, in the raw material leaching process, can bring the big molecular impurity of various plants, as protein, nucleic acid etc.Although the low percentages of protein in the plant is less by the amount of organic solvent extraction, the protein remains of trace is enough to cause bringing out allergic reaction.With the physiognomy ratio, phytoprotein and people's homology difference is big, therefore usually has very strong antigenicity.It is existing that " Chinese pharmacopoeia (version in 2010) control traditional Chinese medicine method for determination of protein has still been continued to use sulfosalicylic acid natural sedimentation method in the past, the protein of control is limited the quantity of and is about about 37～110 μ g/ml, limit the quantity of if traditional Chinese medicine is lower than this, contained protein official method is difficult to detect.Therefore, the protein content traditional Chinese medicine that is lower than 37 μ g/ml will be regarded as " qualified ".And the acid ingredient in the traditional Chinese medicine can disturb sulfosalicylic acid method to detect the result of protein, and this causes the sulfosalicylic acid method range of application limited.And other protein detection method that do not adopted by pharmacopeia as, Kai Shi (Kjeldahl) nitriding, biuret method, Folin-phenol reagent process, direct Coomassie brilliant blue method (Bradford method), ultraviolet absorption method and BCA method etc., but because other compositions of traditional Chinese medicine or intrinsic colour bring serious interference, specificity is relatively poor, sensitivity is relatively poor, is not suitable for the protein limit detection of traditional Chinese medicine.Protein-based impurity belongs to a class impurity, has comprised the protein of heterogeneity or molecular weight, therefore also is difficult to detect with instrument analytical methods such as HPLC and/or mass spectrums.
The trace protein composition that exists in the traditional Chinese medicine has brought safety issue, controls the protein impurities in the traditional Chinese medicine as strict as possible, helps improving the security of traditional Chinese medicine.Therefore it is necessary to set up the high and sensitive traditional Chinese medicine protein detection method of specificity.
Because traditional Chinese medicine all has darker color, composition is various, and protein is a class material, therefore is difficult to improve the sensitivity and the specificity of detection simultaneously with present single method.Therefore the characteristics that The present invention be directed to traditional Chinese medicine are set up, and protein detection is had the characteristics highly sensitive, that antijamming capability is strong.
We had applied for two patents (application number is respectively 201010199693.3 and 201010504574.4) in the past, two kinds of methods that detect the traditional Chinese medicine trace protein have been invented, the sensitivity that detects is about 12 μ g/ml and 0.5 μ g/ml respectively, and sensitivity is respectively that version in 2010 is " about 3 times and 30 times of the Chinese pharmacopoeia method.In this patent, we improve method once more with reference to the principle of preceding two patents of patent, and detection sensitivity is further improved.
Summary of the invention
The present invention utilizes some film the characteristics and the Coomassie brilliant blue of the absorption of protein specific high strength can be set up this method to the characteristics of protein specific colour developing, to improve sensitivity and the specificity that the traditional Chinese medicine trace protein detects.This method may further comprise the steps:
1, the protein adsorption film is installed on the filter;
2, the Chinese medicine injection agent solution is passed through the protein adsorption film;
3, with the solution washing protein adsorption film that contains organic solvent;
4, the protein adsorption film is carried out the Coomassie brilliant blue colour developing.
In above step 1, the film of adsorbed proteins includes but not limited to nitrocellulose filter, nylon membrane, pvdf membrane (Polyvinylidene fluoride, PVDF membrane).
In above step 2, the volume of Chinese medicine injection agent solution is determined as required.
In above step 3, the organic solution concentration of washed protein adsorption film is 0-100%, and organic solvent includes but not limited to methyl alcohol, ethanol, acetonitrile, acetone, chloroform.Adopt the amount of organic solution washing and the number of times of washing not to limit, to remove traditional Chinese medicine true qualities degree of being.
When step 4 was carried out the Coomassie brilliant blue colour developing, the concentration of Coomassie brilliant blue was not limit.Can direct staining; The processing of decolouring after also can dyeing is to reduce the background of film.The traditional Chinese medicine that contains certain protein concentration can demonstrate stable blueness.
The effect that this method is implemented is, the sensitivity of detection and specificity height, and antijamming capability is strong.
The recommendation condition of the inventive method is: the protein adsorption film adopts pvdf membrane, the filter aperture that the protein adsorption film is installed is 2mm, the volume that filters traditional Chinese medicine injection is 0.5-5ml, the organic solution of washed protein adsorption film is methyl alcohol, and coomassie brilliant blue staining adopts 0.25% coomassie brilliant blue staining liquid, and dyeing time is 5 minutes, adopting methyl alcohol-water-glacial acetic acid mixed liquor is destainer, ratio is 4.5: 4.5: 1, decolours each 10 minutes 3 times.
Description of drawings
Fig. 1 sets up protein detection method for adopting pvdf membrane, determine that the coloration result figure of detectability of the present invention: A is installed on the filtration unit that the filter opening diameter is 2mm for the pvdf membrane sheet, filter standard protein BSA (bovine serum albumin(BSA)) 0.5ml of variable concentrations, the spot figure behind methanol wash and coomassie brilliant blue staining; B is that pvdf membrane sheet filter opening diameter is 2mm, filters the standard protein BSA 5ml of variable concentrations, the spot figure behind methanol wash and coomassie brilliant blue staining; The BSA concentration of point 1-10 is respectively 100,000,33,333,11,111,3,704,1,235,412,137,46,15,5ng/ml, and the protein content of some p is zero.
Fig. 2 sets up protein detection method for adopting nitrocellulose filter (NC film), determine that the coloration result figure of detectability of the present invention: A is installed on the filtration unit that the filter opening diameter is 2mm for the NC diaphragm, filter the standard protein BSA 0.5ml of variable concentrations, the spot figure behind methanol wash and coomassie brilliant blue staining; The BSA concentration of point 1-7 is respectively 100,000,33,333,11,111,3,704,1,235,412,137ng/ml, and the protein content of some p is zero.
Fig. 3 detects protein content figure as a result in 80% ethanol extract for the present invention: with 75% alcohol extract BSA, through high speed (15,000g) get the 0.5ml supernatant after centrifugal back 5 minutes, via hole diameter is that the filtration unit of 2mm passes through pvdf membrane with extract, then row coomassie brilliant blue staining after the acetonitrile washing.
Fig. 4 detects the figure as a result of four kinds of traditional Chinese medicine trace proteins: A for the pvdf membrane sheet is installed on the filtration unit that the filter opening diameter is 2mm for the present invention, filter different traditional Chinese medicine injection 0.5ml, the spot figure behind methanol wash and coomassie brilliant blue staining; B is that pvdf membrane sheet filter opening diameter is 2mm, filters different traditional Chinese medicine injection 5ml, the spot figure behind methanol wash and coomassie brilliant blue staining; Q is a qingkailing injections, and D is a danshen injections, and S is a Shuanhuanglian injection, and Dz is the erigeron breviscapus parenteral solution, and p is a blank.
Embodiment
Exemplifying embodiment 1
Set up filtration with pvdf membrane and hold back the method protein detection method, determine detectability
Get 11 15ml test tube numbering 1-10 and p, with acetate buffer (sodium acetate, 4.8g; Glacial acetic acid, 2.3ml; Add tri-distilled water to 200ml, pH 4.5) will to make concentration be 100, the BSA solution 15ml of 000ng/ml is in No. 1 pipe, in the 2-10 pipe, respectively add the 10ml acetate buffer, from No. 1 pipe, get mixing in 5ml solution to 2 pipe, from No. 2 pipes, get mixing in 5ml solution to 3 pipe again, the rest may be inferred to No. 10 pipe, promptly obtains concentration gradient and be one group of standard solution of 3 times, and the concentration of BSA is followed successively by 100 from No. 1 pipe to 10 pipe, 000,33,333,11,111,3,704,1,235,412,137,46,15,5ng/ml, p number pipe be not for containing the blank of BSA.
It is on the filter of 2mm filtering the aperture that pvdf membrane is carried out mark installment, and filter connects vacuum pump.With moistening filtering holes of methyl alcohol 0.1ml and pvdf membrane, then standard protein solution is added on the filter earlier, starts vacuum pump, standard protein liquid passes pvdf membrane from filter opening.After filtrate had been filtered, the position of conversion pvdf membrane was reinstalled on the filter, and is moistening and filter next standard protein quality sample with methyl alcohol once more.The sample of expection is immersed in the methanol solution washing to this film 3 times, each 10 minutes all after the pvdf membrane upward filtration.Afterwards with 0.25% coomassie brilliant blue staining liquid dyeing 5 minutes, shifting on the decolorization swinging table with destainer methyl alcohol-water-glacial acetic acid (4.5: 4.5: 1) 10ml wash-out 3 times, each 5 minutes, slough background colour, film is taken pictures, observe the spot colors power.The result as shown in Figure 1.
From Fig. 1 as seen, when filtering volume and be 0.5ml, nonprotein acetate buffer solution is by behind the pvdf membrane, and coomassie brilliant blue staining is almost colourless, but when filtering volume and being increased to 5ml, the dyeing of this point has intensification slightly.And the filtration spot of acetate buffer solution that contains protein is all dark than blank spot (some p).When the filtration volume was 0.5ml, the protein detection of this law was limited the quantity of at (figure A point 7) below the 137ng/ml; When the filtration volume was increased to 5ml, the detection of this law was limited the quantity of and can be reached 5-15ng/ml.
Exemplifying embodiment 2
Set up filtration with the NC film and hold back the method protein detection method, determine detectability
Get 8 1.5ml test tube numbering 1-8 and p, with acetate buffer (sodium acetate, 4.8g; Glacial acetic acid, 2.3ml; Add tri-distilled water to 200ml, pH 4.5) will to make concentration be that the BSA solution 1.5ml of 100000ng/ml is in No. 1 pipe, in the 2-10 pipe, respectively add the 1ml acetate buffer, from No. 1 pipe, get mixing in 0.5ml solution to 2 pipe, from No. 2 pipes, get mixing in 0.5ml solution to 3 pipe again, the rest may be inferred to No. 10 pipe, promptly obtain concentration gradient and be one group of standard solution of 3 times, the concentration of BSA is followed successively by 100,000,33,333,11 from No. 1 pipe to 7 pipe, 111,3,704,1,235,412,137ng/ml, p number pipe be not for containing the blank of BSA.
It is on the filter of 2mm filtering the aperture that the NC film is carried out mark installment, and filter connects vacuum pump.With moistening filtering holes of methyl alcohol 0.1ml and NC film, then standard protein solution is added on the filter earlier, starts vacuum pump, standard protein liquid passes the NC film from filter opening.After filtrate had been filtered, the position of conversion NC film was reinstalled on the filter, and is moistening and filter next standard protein quality sample with methyl alcohol once more.The sample of expection is immersed in the methanol solution washing to this film 3 times, each 10 minutes all after the NC film upward filtration.Afterwards with 0.25% coomassie brilliant blue staining liquid dyeing 5 minutes, shifting on the decolorization swinging table with destainer methyl alcohol-water-glacial acetic acid (4.5: 4.5: 1) 10ml wash-out 3 times, each 5 minutes, slough background colour, film is taken pictures, observe the spot colors power.The result as shown in Figure 1.
From Fig. 2 as seen, when the filtration volume was 0.5ml, nonprotein acetate buffer solution was by behind the NC film, and coomassie brilliant blue staining is almost colourless.When the filtration volume was 0.5ml, the protein detection of this law was limited the quantity of about (figure A point 6) 412ng/ml.Compare the employing pvdf membrane, the detection effect of NC film is more bad.
Exemplifying embodiment 3
The present invention directly detects the protein in 80% ethanol extract
The raw materials for production of traditional Chinese medicine often adopt " poach alcohol precipitation " to produce, and the concentration of alcohol of precipitation usefulness mostly is 80%, now adopts this law to detect the dissolving situation of 80% ethanol to protein.
Get 1 1.5ml test tube.Take by weighing 18mg BSA as for wherein, add 1ml 80% ethanol shake well.Through 15, centrifugal 5 minutes of 000g gets supernatant 0.5ml afterwards.
It is on the filter of 2mm filtering the aperture that pvdf membrane is carried out mark installment, and filter connects vacuum pump.With moistening filtering holes of methyl alcohol 0.1ml and pvdf membrane, then supernatant 0.5ml is added on the filter earlier, starts vacuum pump, supernatant passes pvdf membrane from filter opening.This film is immersed in the acetonitrile solution washing 3 times, each 10 minutes.Afterwards with 0.25% coomassie brilliant blue staining liquid dyeing 5 minutes, shifting on the decolorization swinging table with destainer methyl alcohol-water-glacial acetic acid (4.5: 4.5: 1) 10ml wash-out 3 times, each 5 minutes, slough background colour, film is taken pictures, observe the spot colors power.The result as shown in Figure 3.
As seen from Figure 3, it is darker to filter spot colors, and comparison diagram 1 infers that protein content in 80% ethanol extract is up to 11-33 μ g/ml.
Exemplifying embodiment 4
Traditional Chinese medicine finished product trace protein detects
Buy qingkailing injections, Shuanhuanglian injection, danshen injections and erigeron breviscapus parenteral solution from the market as test sample.It is on the filter of 2mm filtering the aperture that pvdf membrane is carried out mark installment, and filter connects vacuum pump.With moistening filtering holes of methyl alcohol 0.1ml and pvdf membrane, then traditional Chinese medicine is added on the filter earlier, starts vacuum pump, traditional Chinese medicine injection passes pvdf membrane from filter opening.After filtrate had been filtered, the position of conversion pvdf membrane was reinstalled on the filter, and is moistening and filter next traditional Chinese medicine sample with methyl alcohol once more.Sample is immersed in the methanol solution washing to this film 3 times, each 10 minutes all after the pvdf membrane upward filtration.Afterwards with 0.25% coomassie brilliant blue staining liquid dyeing 5 minutes, shifting on the decolorization swinging table with destainer methyl alcohol-water-glacial acetic acid (4.5: 4.5: 1) 10ml wash-out 3 times, each 5 minutes, slough background colour, film is taken pictures, observe the spot colors power.The result as shown in Figure 4.
From Fig. 4 as seen, no matter filter volume be 0.5ml (Fig. 4 A) still to filter volume be 5ml (Fig. 4 B), all can detect qingkailing injections and have more protein, secondly danshen injections and erigeron breviscapus parenteral solution also can be found trace protein.In these four kinds of injections, the Shuanhuanglian injection protein content is minimum.
Claims (4)
1. method that detects the traditional Chinese medicine trace protein, its feature with carry out according to the following steps:
1) will be installed on the filter on the protein adsorption film earlier;
2) traditional Chinese medicine injection is passed through the protein adsorption film;
3) with the solution washing protein adsorption film that contains organic solvent;
4) the protein adsorption film is carried out coomassie brilliant blue staining.
2. according to the described traditional Chinese medicine trace protein of claim 1 detection method, it is characterized in that the film that is used for adsorbed proteins includes but not limited to pvdf membrane (PVDF membrane), nitrocellulose filter (NC film).
3. according to the described traditional Chinese medicine trace protein of claim 1 detection method, it is characterized in that washing the used organic solution concentration of removal traditional Chinese medicine point sample true qualities is 0-100%, organic solvent includes but not limited to methyl alcohol, acetonitrile, and the mixing of their two or more arbitrary proportions; Adopt the amount of organic solution washing and the number of times of washing not to limit, to remove the true qualities degree of being of traditional Chinese medicine point sample spot.
4. according to the described traditional Chinese medicine trace protein of claim 1 detection method, it is characterized in that the Coomassie brilliant blue concentration that dyes used do not limit; Both can be to the point sample film direct staining after handling through step 2, the processing of decolouring again after also can dyeing is with further reduction dyeing background; What the dyeing rear decoloring adopted is that content is the organic solution of 0-100%, includes but not limited to methyl alcohol, acetonitrile, and the mixing of their two or more arbitrary proportions.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111721884A (en) * | 2020-06-30 | 2020-09-29 | 南京中医药大学 | Method for detecting trace plant protein components in thermite injection and intermediate thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07243972A (en) * | 1994-03-04 | 1995-09-19 | Nippon Oil & Fats Co Ltd | Quantitative determination method for trace protein in lipid |
| CN101865926A (en) * | 2010-06-13 | 2010-10-20 | 云南中医学院 | Detection method for trace protein of Chinese medicinal injection |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07243972A (en) * | 1994-03-04 | 1995-09-19 | Nippon Oil & Fats Co Ltd | Quantitative determination method for trace protein in lipid |
| CN101865926A (en) * | 2010-06-13 | 2010-10-20 | 云南中医学院 | Detection method for trace protein of Chinese medicinal injection |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111721884A (en) * | 2020-06-30 | 2020-09-29 | 南京中医药大学 | Method for detecting trace plant protein components in thermite injection and intermediate thereof |
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