CN103901010A - Biological activity testing method of Chinese medicine preparation containing component of leech or earthworm - Google Patents
Biological activity testing method of Chinese medicine preparation containing component of leech or earthworm Download PDFInfo
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Abstract
The invention discloses a biological activity testing method of a Chinese medicine preparation containing a component of leech or earthworm. In-vitro experiment shows that a leach or earthworm extract liquid can inhibit the thrombin activity in a dose-dependent manner, so that the inhibition function of thrombin can be tested by using a fluorescence resonance energy transfer (FRET) method, the anti-coagulation activity of a medicine preparation containing medicine components such as leach or earthworm can be reflected, and the method is simple, convenient, rapid, small in error and high in reproducibility.
Description
Technical field
The present invention relates to the quality determining method of Chinese medicine preparation, definite says, the present invention relates to a kind of biological activity determination method of the Chinese medicine preparation that contains leech or earthworm medicinal ingredient, and the method can be used for the quality control of product.
Background technology
Leech is traditional simply Chinese medicine, begin to be loaded in Shennong's Herbal, the traditional Chinese medical science think it there is broken blood, by the stasis of blood, the effect of stimulating the menstrual flow.Always be used for the treatment of abdominal mass, lump in the abdomen, blood stasis amenorrhoea, traumatic injury.Hirudin in leech is to act on the strongest thrombin inhibitor, and it can not only stop fibrinogenic solidifying, and also can stop the further blood stasis reaction of catalyzed by thrombin, as the platelet response of blood coagulation induction etc.Can effectively suppress to dissociate due to hirudin with sludged blood on fibrin ferment, therefore can be with the formation to prevent all kinds of thrombus and extension.Studies have shown that, leech medicinal material is after different extraction processes, and its anticoagulating active does not exist only in hirudin, and little point of subconstiuent of part also has anticoagulation, and the Chinese medicine preparation of this constituents is as SHUXUETONG ZHUSHEYE etc.
Earthworm is also traditional Chinese medicine simply, has heat-clearing, arresting convulsion, dredging collateral, relievings asthma and the multiple efficacies such as diuresis.Modern study shows, earthworm contains abundant enzyme, and wherein fibrinoclase, Lumbrokinase, earthworm colloid enzyme have wide influence to body intravascular coagulation and fibrinolytic system.He Shilin etc. (anticoagulation of earthworm extract and toxicity [J]. Hunan Medical University's journal, 1990, (2): 107-111) find all there is anticoagulant active outside earthworm decocting alcohol extract in animal body, think that through preliminary Chemical Decomposition anticoagulating active composition may be heat-resisting small peptide composition, side by side except protein, polysaccharide, the macromolecular possibility of nucleic acid.The Chinese medicine preparation that contains earthworm composition is as clinical controls that is widely used in the diseases such as headstroke such as compound formula earthworm capsule, brain Xintong capsules.
The Chinese medicine preparation that contains leech or earthworm medicinal material has many-sided physiologically actives such as anti-freezing, thrombolysis, cytoprotection more, is used for clinically the control of cardiovascular and cerebrovascular relevant disease.At present, also there is no good biological activity determination method for this class Chinese medicine preparation.State-promulgated pharmacopoeia 2010 editions leech medicinal material anticoagulating active assay methods used adopt fibrin ferment titrimetrys, and timing and naked eyes are differentiated titration end-point, the large and easy subject's operation technique impact of error.The inhibiting effect that the present invention adopts FRET (fluorescence resonance energy transfer) (FRET) method to measure fibrin ferment can reflect the anticoagulating active of the pharmaceutical preparation that contains leech or earthworm medicinal ingredient, adopt luminoscope robot reading, method is easy fast, error is little, reappearance is high, has great advantage compared with official method tool.
Fibrin ferment is the very strong serine stretch protein class hydrolytic enzyme of a species specificity being become by prothrombin activation, is the natural component in body blood coagulation system.Fibrin ferment exists with factor form in vivo, and in coagulation process, prothrombin complex changes its activation into have serine stretch protein class hydrolysing activity fibrin ferment.Central role has been played in being formed on of fibrin ferment in coagulation process.Some peptide class substrates of the specific shearing of fibrin ferment as the existing bibliographical informations such as S-2238 (H-D-Phe-Pip-Arg-pNA2HCl) (Abildgaard U, Lie M,
oR.Antithrombin (heparin cofactor) assay with " new " chromogenic substrates (S-2238and Chromozym TH) .Thromb Res.1977Oct; 11 (4): 549-53.).
Summary of the invention
The deficiency that the object of the invention is to overcome the existing Chinese medicine preparation biological activity determination method containing leech or earthworm medicinal material, provides a kind of new biologically active detection method, to control the quality of product.
Mensuration provided by the invention, containing the Chinese medicine preparation antithrombase biological activity determination method of leech or earthworm medicinal material, mainly comprises the following steps:
(1) formulated need testing solution: for injection, can directly get parenteral solution as need testing solution, powder ampoule agent for injection can adopt after physiological saline solution as need testing solution, solid pharmaceutical preparation can adopt proper method to extract after active component, after water, physiological saline or damping fluid dissolve as need testing solution.
(2) preparation feedback solution: add successively assay buffer, physiological saline, need testing solution, the fibrin ferment of 37 DEG C of preheatings in 96 hole fluorometric assay plates, mix and obtain reaction solution.
(3) measure fluorescence intensity: by above-mentioned 37 DEG C of preheating 15min of 96 hole fluorescent plate that added solution, add fibrin ferment substrate solution 50 μ l, Ex/Em=485nm/535nm measures fluorescence intensity (RFU) immediately, measures and continues 30min, at interval of 5min record data.
In described step (1), the test sample compound method of solid pharmaceutical preparation is that precision takes solid pharmaceutical preparation 0.1～10g, adopt water, physiological saline or methyl alcohol, ethanolic solution to extract, extracting mode can adopt immersion, heating to extract or ultrasonic extraction, extract is evaporated to dry, after water, physiological saline or Tris damping fluid dissolve as need testing solution.
In described step (2), the preparation method of reaction solution is that every hole adds need testing solution 10～90 μ l in 96 hole fluorometric assay plates, fibrin ferment 20～200ng or the 5mU～5U of purifying, and assay buffer or physiological saline are to cumulative volume 100 μ l.Assay buffer can adopt the 0.01～5M Tris damping fluid within the scope of pH6-8.
In described step (3), fibrin ferment substrate is fluorescently-labeled little peptide, fluorescent marker can be the fluorescent dyes such as biotin or FITC, little peptide core amino acid sequence is other sequences of Phe-Pro-Arg or bibliographical information, and the concentration of fluorogenic substrate is 0.1-100 μ M.
FRET (fluorescence resonance energy transfer) (FRET) method detects principle and sees accompanying drawing 1, fibrin ferment energy specificity is sheared thrombin peptide class substrate, peptide class substrate General N end and C section have respectively a donor and acceptor fluorescence group, under normal condition, donor acceptor groups is on same peptide chain, fluorescent quenching occurs because the two is near each other, and donor does not have fluorescence to produce.When adding after fibrin ferment, can specificity cut off peptide class substrate, fluorescent quenching stops, and in the time having external source exciting light, donor fluorophor can produce fluorescence.The power of FRET fluorescence is directly proportional to thrombin activity, and thrombin activity is higher, and fluorescence is stronger.Fluorescence reading with the blank solvent that adds/do not add fibrin ferment is respectively RFU (2) and RFU (1), the fluorescence reading that adds/do not add the test solution of fibrin ferment is respectively RFU (4) and RFU (3), and antithrombin activity can be calculated as follows:
Brief description of the drawings
Accompanying drawing 1: FRET (fluorescence resonance energy transfer) (FRET) principle schematic
The fluorescence numerical value of accompanying drawing 2: enzymatic activity RFU (2)-RFU (1) and reaction time (min) linear schematic diagram
Accompanying drawing 3: hirudin amount effect relation curve figure
Embodiment
Following examples are in order to the present invention to be described, but are not used for limiting the scope of the invention.SHUXUETONG ZHUSHEYE in following examples is purchased from Mudanjiang Youbo Pharmaceutical Co., Ltd., the proportioning of this parenteral solution and preparation method are shown in the embodiment 1 of Granted publication CN1192782C, kit and fluorogenic substrate H-D-Phe-Pro-Arg-AFC are purchased from AnaSpec company of the U.S., fibrin ferment is examined and determine institute purchased from Chinese pharmaceutical biological product, lepirudin 023 ludon is purchased from Millipore company of the U.S., EnSpire
tMmultilabel Plate Reader microwell plate detector is Perkin-Elmer company product, and other reagent are pure for analyzing.
Embodiment 1: thrombin activity is investigated with adding reaction time relation after substrate
Adopt AnaSpec company of the U.S.
520 kits, test by following flow process:
(1) preparation work liquid
1 × assay buffer: 2 × assay buffer of 5ml (reagent C) is added in 5ml deionized water;
Fibrin ferment substrate solution: use 1 × assay buffer to carry out 100 times of dilutions to fibrin ferment substrate (reagent A), each test all needs to prepare fresh substrate solution, because the fibrin ferment substrate in reagent A, to photaesthesia, please lucifuge operate;
0.9% normal saline solution: dissolve 0.9g NaCl in 100ml deionized water;
(2) preparation feedback measured in solution fluorescence intensity
According to the form below adds 1 × assay buffer, 0.9% physiological saline, the fibrin ferment of 37 DEG C of preheatings successively in 96 hole fluorometric assay plates, mixes and obtain reaction solution;
Table 1 thrombin activity assay process table
The fluorescence numerical value of RFU (2)-RFU (1) can represent thrombin activity.
Result: fluorescence intensity data are as table 2, RFU (2)-RFU (1) is the linear mapping of minute with the enzyme reaction time, sees that accompanying drawing 2 least square methods try to achieve equation of linear regression.Equation is Y=132480X+194993, coefficient R
2=0.9982, show that enzymatic activity and reaction time present good linear relationship within the scope of 0～30min.
Table 2 fluorescence reading and minute relation table
Embodiment 2: the antithrombase effect of hirudin is investigated
(1) preparation work liquid
Assay buffer: 0.04M Tris-HCl damping fluid (pH7.8): get Tris2.42g, sodium chloride 4.06g, the 400ml that adds water dissolves, and adjusts pH to 7.8 with 4mol/L HCl solution, adds water to 500ml;
Fibrin ferment substrate solution: use test damping fluid carries out 100 times of dilutions to fibrin ferment fluorogenic substrate H-D-Phe-Pro-Arg-AFC (AnaSpec company of the U.S.), each test all needs to prepare fresh substrate solution, because fibrin ferment substrate, to photaesthesia, please lucifuge operate;
Normal saline solution: dissolve 0.9g NaCl in 100ml deionized water;
Hirudin solution: adopt assay buffer dilution hirudin to desired concn.
(2) preparation feedback measured in solution fluorescence intensity
Press method in embodiment 1, add successively assay buffer, hirudin, physiological saline, the fibrin ferment of 37 DEG C of preheatings in 96 hole fluorometric assay plates, (cumulative volume 50 μ l) to mix and obtain reaction solution.
(3) measure
37 DEG C of preheating microwell plate 15min, open EnSpire
tMmultilabel Plate Reader microwell plate detector, starts EnVision Workstation version1.12 program, and design temperature is 37 DEG C.In 96 hole fluorometric assay plates, every hole adds fibrin ferment fluorogenic substrate solution 50 μ l, and Ex/Em=485nm/535nm measures fluorescence intensity (RFU) immediately, measures and continues 30min, at interval of 5min record data.
Result: the live vol effect relationship of hirudin Trombin inhibiting is shown in accompanying drawing 3, visible hirudin is the Trombin inhibiting activity of dose dependent, IC50=3.1nM.
Embodiment 3: the antithrombase effect of SHUXUETONG ZHUSHEYE is investigated
Adopt AnaSpec company of the U.S.
520 kits, test by following flow process.
(1) preparation work liquid
1 × assay buffer: 2 × assay buffer of 5ml is added in 5ml deionized water;
Fibrin ferment substrate solution: use 1 × assay buffer to carry out 100 times of dilutions to fibrin ferment substrate, each test all needs to prepare fresh substrate solution, because the fibrin ferment substrate in reagent A, to photaesthesia, please lucifuge operate;
Normal saline solution: dissolve 0.9g NaCl in 100ml deionized water;
SHUXUETONG ZHUSHEYE: Mudanjiang Youbo Pharmaceutical Co., Ltd. provides, lot number 11111311,11112011,11112512.
(2) preparation feedback solution
According to the form below in 96 hole fluorometric assay plates, add successively 37 DEG C of preheatings 1 × assay buffer, SHUXUETONG ZHUSHEYE (every hole 32 μ l), physiological saline, fibrin ferment (every hole 50mU), (cumulative volume 50 μ are l) to mix and obtain reaction solution.
(3) measure
37 DEG C of preheating microwell plate 15min, open EnSpire
tMmultilabel Plate Reader microwell plate detector, starts EnVision Workstation version1.12 program, and design temperature is 37 DEG C.In 96 hole fluorometric assay plates, every hole adds fibrin ferment fluorogenic substrate solution 50 μ l, and Ex/Em=485nm/535nm measures fluorescence intensity (RFU) immediately, measures and continues 30min, at interval of 5min record data.
Fluorescence reading with the blank solvent that adds/do not add fibrin ferment is respectively RFU (2) and RFU (1), the fluorescence reading that adds/do not add the test solution of fibrin ferment is respectively RFU (4) and RFU (3), and antithrombin activity can be calculated as follows:
Antithrombin activity (%) calculates as table 3:
Table 3 different batches SHUXUETONG ZHUSHEYE antithrombin activity table
Visible, adding fluorogenic substrate, i.e. enzyme reaction starts in 15～30min, and it is active more stable that SHUXUETONG ZHUSHEYE suppresses, and in 3 batches of products, lot number 11112512 activity are the highest.
Embodiment 4: the antithrombase effect of brain Xintong capsule is investigated
Adopt AnaSpec company of the U.S.
520 kits, test by following flow process:
(1) preparation work liquid
1 × assay buffer: 2 × assay buffer of 5ml is added in 5ml deionized water;
Fibrin ferment substrate solution: use 1 × assay buffer to carry out 100 times of dilutions to fibrin ferment substrate, each test all needs to prepare fresh substrate solution, because the fibrin ferment substrate in reagent A, to photaesthesia, please lucifuge operate;
Normal saline solution: dissolve 0.9g NaCl in 100ml deionized water.
Brain Xintong capsule is provided by Shaanxi Buchang Pharmaceuticals Co., Ltd., lot number 100941.Test liquid preparation method is as follows: get 20 of capsules, incline and content and weigh.Get content 2g, with the ultrasonic extraction of 10% ethanol 30ml 3 times, each 10min, merges extract, 40 DEG C of drying under reduced pressure, and residue is brain Xintong capsule test liquid with 5ml physiological saline solution.
(2) preparation feedback solution
According to the form below in 96 hole fluorometric assay plates, add successively 37 DEG C of preheatings 1 × assay buffer, brain Xintong capsule test liquid (every hole 32 μ l), physiological saline, fibrin ferment (every hole 50mU), (cumulative volume 50 μ are l) to mix and obtain reaction solution.
(3) measure
37 DEG C of preheating microwell plate 15min, open EnSpire
tMmultilabel Plate Reader microwell plate detector, starts EnVision Workstation version1.12 program, and design temperature is 37 DEG C.In 96 hole fluorometric assay plates, every hole adds fibrin ferment fluorogenic substrate solution 50 μ l, and Ex/Em=485nm/535nm measures fluorescence intensity (RFU) immediately, measures and continues 30min, at interval of 5min record data.
Fluorescence reading with the blank solvent that adds/do not add fibrin ferment is respectively RFU (2) and RFU (1), the fluorescence reading that adds/do not add the test solution of fibrin ferment is respectively RFU (4) and RFU (3), and antithrombin activity can be calculated as follows:
Antithrombin activity (%) calculates as table 4:
Table 4 different batches brain Xintong capsule antithrombin activity table
Visible, adding fluorogenic substrate, i.e. enzyme reaction starts in 15～30min, and the logical test liquid of the brain heart suppresses active more stable, and in 3 batches of products, lot number 100941 activity are the highest.
Embodiment 5: the antithrombase effect of day imperial capsule for freeing collateral vessels is investigated
Adopt AnaSpec company of the U.S.
520 kits, test by following flow process:
(1) preparation work liquid
1 × assay buffer: 2 × assay buffer of 5ml is added in 5ml deionized water;
Fibrin ferment substrate solution: use 1 × assay buffer to carry out 100 times of dilutions to fibrin ferment substrate, each test all needs to prepare fresh substrate solution, because the fibrin ferment substrate in reagent A, to photaesthesia, please lucifuge operate;
Normal saline solution: dissolve 0.9g NaCl in 100ml deionized water;
It imperial capsule for freeing collateral vessels: Mudanjiang Youbo Pharmaceutical Co., Ltd. provides, lot number 111001,111002,111003.Test liquid preparation method is as follows: get 20 of capsules, incline and content and weigh.Get content 2g, with the ultrasonic extraction of 10% ethanol 30ml 3 times, each 10min, merges extract, 40 DEG C of drying under reduced pressure, and residue is a day imperial capsule for freeing collateral vessels test liquid with 5ml physiological saline solution.
(2) preparation feedback solution
According to the form below in 96 hole fluorometric assay plates, add successively 37 DEG C of preheatings 1 × assay buffer, day imperial capsule for freeing collateral vessels test liquid (every hole 32 μ l), physiological saline, fibrin ferment (every hole 50mU), (cumulative volume 50 μ are l) to mix and obtain reaction solution.
(3) measure
37 DEG C of preheating microwell plate 15min, open EnSpire
tMmultilabel Plate Reader microwell plate detector, starts EnVision Workstation version1.12 program, and design temperature is 37 DEG C.In 96 hole fluorometric assay plates, every hole adds fibrin ferment fluorogenic substrate solution 50 μ l, and Ex/Em=485nm/535nm measures fluorescence intensity (RFU) immediately, measures and continues 30min, at interval of 5min record data.
Fluorescence reading with the blank solvent that adds/do not add fibrin ferment is respectively RFU (2) and RFU (1), the fluorescence reading that adds/do not add the test solution of fibrin ferment is respectively RFU (4) and RFU (3), and antithrombin activity can be calculated as follows:
Antithrombin activity (%) calculates as table 5:
Table 5 different batches sky dragon capsule for freeing collateral vessels antithrombin activity table
Visible, adding fluorogenic substrate, i.e. enzyme reaction starts in 15～30min, and it is active more stable that a day imperial capsule for freeing collateral vessels test liquid suppresses, and in 3 batches of products, lot number 111001 activity are the highest.
Claims (7)
1. a biologically active detection method that contains the Chinese medicine preparation of leech or earthworm composition, is characterized in that, by detecting the anti-freezing biologically active of active reaction said preparation of fibrin ferment.
2. biological activity determination method according to claim 1, it is characterized in that, detect thrombin activity size indirectly to reflect the antithrombase effect of the Chinese medicine preparation that contains leech or earthworm composition by FRET (fluorescence resonance energy transfer) (FRET) method.
3. biological activity determination method according to claim 1, is characterized in that, adopts the peptide class substrate of fluorescently-labeled fibrin ferment, reflects the activity of fibrin ferment by monitoring the variation of its fluorescence intensity.
4. biological activity determination method according to claim 2, is characterized in that, FRET (fluorescence resonance energy transfer) (FRET) the method concrete operation step of described detection thrombin activity is as follows:
AnaSpec company of the employing U.S.
520 kits
(1) preparation work liquid
1 × assay buffer: 2 × assay buffer of 5ml (reagent C) is added in 5ml deionized water;
Fibrin ferment dilution: use 1 × assay buffer to carry out 25 times of dilutions to fibrin ferment (reagent D), 96 orifice plates, every hole adds the thrombin solution 5 μ l after dilution, adjusts as required the enzyme amount of diluting;
Fibrin ferment substrate solution: use 1 × assay buffer to carry out 100 times of dilutions to fibrin ferment substrate (reagent A), each test all needs to prepare fresh substrate solution, because the fibrin ferment substrate in reagent A, to photaesthesia, please lucifuge operate;
0.9% normal saline solution: dissolve 0.9g NaCl in 100ml deionized water;
(2) preparation feedback solution
In 96 hole fluorometric assay plates, add successively 1 × assay buffer, 0.9% physiological saline of 37 DEG C of preheatings, Chinese medicine extract, the fibrin ferment that contains leech or earthworm medicinal ingredient, mix and obtain reaction solution;
(3) measure fluorescence intensity
By above-mentioned 37 DEG C of preheating 15min of 96 hole fluorescent plate that added solution, add fibrin ferment substrate solution 50 μ l, Ex/Em=485nm/535nm measures fluorescence intensity (RFU) immediately, measures and continues 30min, at interval of 5min record data.
5. biological activity determination method according to claim 3, is characterized in that, peptide class substrate core amino acid sequence is Phe-Pro-Arg.
6. biological activity determination method according to claim 4, is characterized in that, fibrin ferment 20～200ng that in 96 orifice plates, purifying is contained in every hole, or 5mU～5U.
7. biological activity determination method according to claim 4, in 96 orifice plates, in every 100 μ l reaction solutions, adding the Chinese medicine preparation extracting liquid volume that contains leech or earthworm medicinal ingredient is 10～90 μ l.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1181501A (en) * | 1996-10-30 | 1998-05-13 | 杨祺 | Quantitative fluorescence analysis method for determining biological active substances in various kinds of trace samplings |
| JP2003528320A (en) * | 2000-03-23 | 2003-09-24 | アクシオム バイオテクノロジーズ，インコーポレイテッド | How to record transmembrane potential |
| CN1444033A (en) * | 2002-03-10 | 2003-09-24 | 徐永源 | Visual fluorescence immunoassay method for determining biological active substance |
| CN101477049A (en) * | 2008-12-31 | 2009-07-08 | 中国人民解放军第三○二医院 | Method for detecting anti-influenza virus activity of Chinese medicinal material |
-
2014
- 2014-04-21 CN CN201410162027.0A patent/CN103901010A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1181501A (en) * | 1996-10-30 | 1998-05-13 | 杨祺 | Quantitative fluorescence analysis method for determining biological active substances in various kinds of trace samplings |
| JP2003528320A (en) * | 2000-03-23 | 2003-09-24 | アクシオム バイオテクノロジーズ，インコーポレイテッド | How to record transmembrane potential |
| CN1444033A (en) * | 2002-03-10 | 2003-09-24 | 徐永源 | Visual fluorescence immunoassay method for determining biological active substance |
| CN101477049A (en) * | 2008-12-31 | 2009-07-08 | 中国人民解放军第三○二医院 | Method for detecting anti-influenza virus activity of Chinese medicinal material |
Non-Patent Citations (6)
| Title |
|---|
| ANASPEC: "SensoLyte® 520 Thrombin Activity Assay Kit *Fluorimetric*", 《ANASPEC》 * |
| 上海经科化学科技有限公司: ""原装进口凝血酶活性荧光检测试剂盒SensoLyte®520"", 《HTTP://WWW.LABBASE.NET/SUPPLY/SUPPLYITEMS-3134083.HTML》 * |
| 吴志军 等: ""四种不同品种水蛭生物活性的研究与比较"", 《中成药》 * |
| 王兰英等: "《当代医学新进展》", 31 December 1996 * |
| 王晓梅 等: ""荧光共振能量转移技术及其在药物定量分析中的应用"", 《药学进展》 * |
| 胡醒 等: ""BAEE 法测定重组水蛭素的生物活性"", 《中国生化药物杂志》 * |
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Address after: 157013 Heilongjiang province Mudanjiang City Yangming District Yumin Road No. 288 Applicant after: Mudanjiang Youbo Pharmaceutical Co.,Ltd. Address before: 157013 Heilongjiang province Mudanjiang City Yangming District Yumin Road No. 288 Applicant before: Mudanjiang Youbo Pharmaceutical Co., Ltd. |
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