CN104337841A - Anticoagulant fibrinolytic composition in periostracum cicadae - Google Patents
Anticoagulant fibrinolytic composition in periostracum cicadae Download PDFInfo
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Abstract
The invention discloses an anticoagulant fibrinolytic composition in periostracum cicadae, and belongs to the field of traditional Chinese medicines. The anticoagulant fibrinolytic composition in periostracum cicadae is obtained through a separation purification method, and the method concretely comprises the following steps: A, extraction, namely, weighing a periostracum cicadae medicinal material fine powder, adding distilled water, refluxing, extracting, filtering, merging the extraction liquids, concentrating, adding ethanol with the concentration of 95%, standing for a night, performing pumping filtration, and drying the residue, so as to obtain the periostracum cicadae water-extraction alcohol-precipitation substance, then adding distilled water to prepare a solution with the concentration of 10 mg/mL, centrifuging, and getting the supernatant, so as to obtain a loading liquid; and B, purification, namely, weighing macroporous adsorption resin, applying the liquid obtained in the step A to the adsorption resin, performing water eluting, collecting the water eluate, then using ethanol for eluting, and volatilizing the eluate by employing water bath, so as to obtain the anticoagulant fibrinolytic composition in periostracum cicadae. The method is simple in operation, and the obtained target object is relatively high in purity and has good effect when being applied to anticoagulation.
Description
Technical field
The invention belongs to the field of Chinese medicines, be specifically related to traditional Chinese medicine extyaction field.
Background technology
Periostracum Cicadae be cicada Cryptotympana atrata Fabr. (
cryptotympana pustulatafabricius) when nymph sprouts wings, exuviae shell, has dispelling wind and heat pathogens, relieving sore throat to recover voice, the effects such as endogenous wind stopping relieving convulsion, mainly containing compositions such as chitin, protein, organic acid.Modern pharmacology research shows, Periostracum Cicadae, except having antipyretic, analgesia, calmness, convulsion, spasmolytic etc. and acting on, also has certain anticoagulant fibrinolysis activity, but current little to the research on its anticoagulant fibrinolytic active substance basis.
Because macroporous adsorbent resin is a kind of porous macromolecular material, there is porous and larger specific surface area, by the effect such as physical absorption adsorb organic compound matter selectively from solution, thus reach the object of separation and purification, and in Separation of Natural Products, purification etc., demonstrate great advantage.Compared with polydextran gel purification technique, the advantages such as it is low that macroporous adsorbent resin has cost, simple and easy to do, macroporous adsorbent resin has many types, needs research to seek more simple and feasible process in the active substance basis of separation and purification medicine.
Summary of the invention
The object of the invention is to provide anticoagulant fibrinolysis component in a kind of Periostracum Cicadae.
The technical solution adopted in the present invention is:
Anticoagulant fibrinolysis component in a kind of Periostracum Cicadae, its isolation and purification method is made up of following steps:
A, extraction: take Periostracum Cicadae fine medicinal material powder, add distilled water, reflux, extract, 3 times, each 1h, filters, merge extractive liquid, be concentrated into medicinal liquid than being 1:1, adding 95% ethanol to alcohol content is 90%, places sucking filtration after spending the night, residue is dry, and obtain Periostracum Cicadae water extracting alcohol hypostasis, then adding distil water is mixed with concentration is 10mg/ml solution, ultrasonic 10min, centrifugal, get supernatant, obtain sample solution;
B, purification: take macroporous adsorbent resin, blade diameter length ratio is 1:8-12, and loading flow velocity is 2BVh
-1, loading volume is 15-35ml, collects post liquid, then carries out eluting with the 10-50% ethanol of 5-6BV, collects eluent, eluent water-bath is volatilized, to obtain final product.
Preferably, in described step B, blade diameter length ratio is 1:12.
Preferably, in described step B, loading volume is 25ml.
Preferably, in described step B, ethanol is 50%.
Preferably, in described step B, macroporous adsorbent resin is NKA-9 type resin.
As optimal way, described method is made up of following steps:
A, extraction: take Periostracum Cicadae fine medicinal material powder, add distilled water, reflux, extract, 3 times, each 1h, filters, merge extractive liquid, be concentrated into medicinal liquid than being 1:1, adding 95% ethanol to alcohol content is 90%, places sucking filtration after after spending the night, residue is dry, and obtain Periostracum Cicadae water extracting alcohol hypostasis, then adding distil water is mixed with concentration is 10mg/ml solution, ultrasonic 10min, centrifugal, get supernatant, obtain sample solution;
B, purification: take NKA-9 type resin, blade diameter length ratio is 1:12, and loading flow velocity is 2BVh
-1, loading volume is 25ml, collects post liquid, then carries out eluting with 50% ethanol of 5BV, collects eluent, eluent water-bath is volatilized, to obtain final product.
For verifying that the feasibility of the method has done following experiment
1 instrument and reagent
1.1 instrumentlG-PABER-I type platelet aggregation instrument (Beijing Zhong Qinshidi scientific instrument company limited); UV-2000 type spectrophotometer (Shanghai Xianke Instrument Co., Ltd.); 800B type centrifuge (Anting Scientific Instrument Factory, Shanghai); ES-120 type electronic analytical balance (Changsha Xiang Ping development in science and technology company limited); DZ-1BC type vacuum drying oven (Tianjin Stettlen Instrument Ltd.); MVS-1 type turbula shaker (Beijing Jin Bei moral Trade Co., Ltd.); NKA-9 type macroporous adsorbent resin (Tianjin sea light Chemical Co., Ltd.).
reagentperiostracum Cicadae (Yi Ling limited company provides by Shijiazhuang); PT test kit (Shanghai sun biological reagent company); APTT test kit (Shanghai sun biological reagent company); Protein Assay Dye Reagent (Bio-red 5000006, the flat science and technology limited Company in pool, Beijing); Bovine serum albumin (BSA, Sigma A7906, Beijing ring Ya Taike biomedical technology company limited); Ethanol (analytical pure); Urethane (Chemical Reagent Co., Ltd., Sinopharm Group, analytical pure); Sodium citrate (Xilong Chemical Co., Ltd, analytical pure); Calcium chloride (Xilong Chemical Co., Ltd, analytical pure); Sodium chloride (Xilong Chemical Co., Ltd, analytical pure).
animalhealthy rabbits (male), purchased from the prosperous laboratory animal plant in Haidian, Beijing, weight is about 2kg, animal credit number: SCXK (capital) 2011-0006.
method and result
2.1 test sample preparations
2.1.1 reference substance solutionprecision takes bovine serum albumin 10mg, and adding appropriate distilled water, to be mixed with concentration be 1mg/ml solution, is reference substance solution.
periostracum Cicadae water extracting alcohol hypostasistake Periostracum Cicadae fine medicinal material powder 150g and be placed in 5000ml round-bottomed flask, add 3000ml distilled water, reflux, extract, 3 times, each 1h, filters, merge extractive liquid, be concentrated into medicinal liquid than 1:1, adding 95% ethanol to alcohol content is 90%, put in refrigerator place spend the night after sucking filtration, residue is dry, obtains Periostracum Cicadae water extracting alcohol hypostasis.
macroporous adsorbent resin sample solutionget Periostracum Cicadae water extracting alcohol hypostasis appropriate, adding appropriate distilled water, to be mixed with concentration be 10mg/ml solution, and ultrasonic 10min is centrifugal, gets supernatant, is load solution.
evaluation of pesticide effectiveness sampleafter getting macroporous adsorbent resin eluting, gained object is appropriate, adds normal saline and becomes concentration to be 10mg/ ml solution, be evaluation of pesticide effectiveness solution.
content assaying method
2.2.1 the selection of maximum absorption wavelengthprecision measures reference substance solution and each 0.1 mL of sample solution, add 5ml coomassie brilliant blue staining liquid respectively, leave standstill 15 min after mixing, scan in 200 ~ 900 nm wave-length coverages, both results all have absorption maximum at 588nm place, and negative control is noiseless.
specification Curve of Increasingprecision measure reference substance solution 0.02,0.04,0.06,0.08,0.1ml in 10ml test tube, mend to 0.1ml with distilled water successively, obtain concentration be 0.2,0.4,0.6,0.8, the BSA solution of 1.0mg/ml, for subsequent use.In each test tube, add 5ml coomassie brilliant blue staining liquid respectively, leave standstill 15 min after mixing, putting 588nm place and measure absorbance, take distilled water as blank.With bovine serum albumin concentration (mg/ml) be abscissa, absorbance (A) for vertical coordinate drawing standard curve, obtaining regression equation is y=1.04x-0.0198, r=0.9980.
precision testaccurate absorption sample solution 0.5mL, replication 6 times, absorbance values is 0.733, RSD=0.14%, shows that instrument precision is good.
stability testget same sample solution to develop the color as stated above, in 0,1,2,3,6,12h measures 6 absorbances, the RSD=2.31% of absorbance altogether, shows that need testing solution is basicly stable in 12h.
replica testget same batch of Periostracum Cicadae fine medicinal material powder 6 parts, make sample solution by 2.1.2 and measure, the meansigma methods calculating protein content in crude drug is 9.15mg/g, RSD=1.90%, shows that this law repeatability is good.
average recovery is testedtake same batch of Periostracum Cicadae fine medicinal material powder 6 parts, every part of 1.5g, precision adds reference substance solution 10 mL, makes solution by 2.1.2, and recording mean sample recovery rate is 96.28%, RSD=1.16%, meets the requirements.
the anticoagulant fibrinolytic test of pesticide effectiveness
2.3.1 the preparation of blood plasmaget the anesthesia of male and healthy rabbit auricular vein injection urethane, carotid artery gets blood, 3.8% sodium citrate anticoagulant (1:9), with the centrifugal 10min of 1000r/min after mixing, get supernatant, obtain platelet rich plasma (PRP), with the centrifugal 10min of 3000r/min, get supernatant, obtain platelet poor plasma (PPP).
testget PPP 100 μ L in test cup, add evaluation of pesticide effectiveness solution 10 μ L, 37 DEG C of pre-temperature 3min, add PT reagent 200 μ L (pre-temperature 3min), start timing, to there being fiber protein yarn to occur, accurate recording setting time.Take normal saline as negative control (each sample parallel measures 6 parts).
testget PPP100 μ L in test cup, add evaluation of pesticide effectiveness solution 10 μ L, 37 DEG C of pre-temperature 3min, add APTT reagent 100 μ L (pre-temperature 5min), after hatching 5min, add CaCl
2100 μ L (pre-temperature 15min), start timing, to there being fiber protein yarn to occur, and accurate recording setting time.Take normal saline as negative control (each sample parallel measures 6 parts).
blood clotting-fibrinolytic Dynamic Graph testget PRP 150 μ L and evaluation of pesticide effectiveness solution 10 μ L mixes, 37 DEG C of pre-temperature 10min, PPP zeroings, add 0.1mol/L CaCl simultaneously
250 μ L, balance recorder record Dynamic Graph, till figure peak follow stablizes 2min, curve obtained is blood clotting-fibrinolytic Dynamic Graph.Following parameter is observed according to Dynamic Graph:
the solidification-stable time (CST)
setting time (CT)
maximum coagulation grade (MCE)
average setting rate (ACR).With normal saline negative control (each sample parallel measures 6 parts).
Result display anticoagulant fibrinolysis component can extend PT, APTT, has anticoagulant effect.
purification with macroreticular resin technical study
2.4.1 resin pretreatmentmacroporous adsorbent resin 95% soak with ethanol 24h, fully swelling, be washed till effluent with ethanol and add appropriate distilled water without till during white opacity phenomenon, be finally washed till without alcohol taste with distilled water, for subsequent use.
sample solution concentration is investigatedget the NKA-9 type resin wet method handled well in right amount and be loaded on (blade diameter length ratio is 1:10) in 1.5cm × 30cm chromatographic column.Take Periostracum Cicadae water extracting alcohol hypostasis 150mg be mixed with respectively concentration be 5,7.5,10,12.5, the sample solution of 15mg/ml, with 2BVh
-1flow velocity absorption after, collected post liquid, after a certain amount of washing, collected water lotion, then carried out eluting with 6BV 50% ethanol, collection eluent, put the absorbance that 588nm place measured post liquid, water lotion and eluent, be calculated as follows resin absorption ration and desorption quantity.After the water-bath of resin elution liquid being volatilized, prepare sample by 2.1.3, measure PT and APTT, the results are shown in Table 1.Result shows, along with the increase of sample solution concentration, absorption ration increases gradually, and desorption efficiency declines gradually.Only sample solution concentration is that the eluate of 10 mg/ml can extend APTT and PT simultaneously, therefore determines that sample solution concentration is 10 mg/ml.
Absorption ration (mg/ml resin)=[M-(M
cross-M
water)]/W
Desorption efficiency (%)=M
alcohol/ [M-(M
cross-M
water)] × 100%
Wherein: M is total protein quality (mg) in sample solution; M
crossfor crossing total protein quality (mg) in post liquid; M
waterfor total protein quality (mg) in water lotion; M
alcoholfor total protein quality (mg) in ethanol elution; W is the volume (ml) of resin.
leakage curve is investigatedmeasure the NKA-9 type resin wet method handled well in right amount and be loaded on (blade diameter length ratio is 1:10) in 1.5cm × 30cm chromatographic column.Getting concentration is that 10mg/ml sample solution 90ml is with 2BVh
-1flow velocity continue through resin column, often 5ml effluent collected by pipe.Put 588nm place and measure effluent absorbance, calculate leakage rate.With loading volume (ml) for abscissa, leakage rate (%) is vertical coordinate, draws and reveals curve.The results are shown in Figure 1.From curve, when loading volume is 40mL, leakage rate increases severely, and illustrates that now sample solution starts obvious leakage.
best applied sample amount investigation amountget the NKA-9 type resin wet method handled well in right amount and be loaded on (blade diameter length ratio is 1:10) in 1.5cm × 30cm chromatographic column.Getting concentration is respectively that 10mg/ml sample solution 15 ml, 25 ml, 35 ml are with 2BVh
-1flow velocity continue through resin column, collected post liquid, after a certain amount of washing, collected water lotion, eluting is carried out again with the ethanol of 6BV 50%, collect eluent, put the absorbance that 588nm place measured post liquid, water lotion and eluent, calculate resin absorption ration and desorption quantity.After the water-bath of resin elution liquid being volatilized, prepare sample by 2.1.3, measure PT and APTT, the results are shown in Table 2.Result shows, along with the increase of applied sample amount, absorption ration and desorption efficiency increase thereupon; Different applied sample amount eluate all can extend APTT, PT, and when applied sample amount is 25mL, effect is the most remarkable.Consider that applied sample amount is too small and can waste resin, excessive then easily impurity is too much, is separated not exclusively, therefore is 25mL in conjunction with test of pesticide effectiveness result determination applied sample amount.
blade diameter length ratio is investigatedmeasure the NKA-9 type resin wet method handled well to be in right amount loaded in 1.5cm × 30cm chromatographic column.Resin column blade diameter length ratio is made to be respectively 1:8,1:10,1:12.Getting concentration is that 10mg/ml sample solution 25ml is with 2BVh
-1flow velocity continue through resin column.Collect post liquid, after a certain amount of washing, collected water lotion, then carried out eluting with 6BV 50% ethanol, and collected eluent, put the absorbance that 588nm place measured post liquid, water lotion and eluent, calculate resin absorption ration and desorption quantity.After the water-bath of resin elution liquid being volatilized, prepare sample by 2.1.3, measure PT and APTT, the results are shown in Table 3.Result shows, different blade diameter length ratio eluate all can significant prolongation PT and APTT, and blade diameter length ratio is that the eluate absorption ration of 1:12 and desorption efficiency are the highest, therefore determines that blade diameter length ratio is 1:12.
eluant strength is investigatedmeasure the NKA-9 type macroporous adsorbent resin dress post handled well in right amount, by optimal adsorption technological operation, collected post liquid, and after a certain amount of washing, collected water lotion, and used each 6 BV of the ethanol of 10%, 30%, 50%, 70%, 90% with 2BVh respectively
-1flow velocity carry out gradient elution, Fractional Collections eluent, put 588nm place and measure eluent absorbance, calculate desorption quantity and desorption quantity.After the water-bath of resin elution liquid being volatilized, prepare sample by 2.1.3, measure PT and APTT, the results are shown in Table 4 and Fig. 2.Result shows, macroporous adsorbent resin first increases rear reduction to the desorption efficiency of Periostracum Cicadae water extracting alcohol hypostasis with the increase of concentration of alcohol, when eluant concentration of alcohol is 50%, its desorption efficiency is maximum reaches 48.24%, 10%, 30%, 50% ethanol elution thing all can significant prolongation APTT, PT, it is not remarkable that 70% ethanol elution thing extends PT effect, and do not show prolongation APTT effect, therefore determine that eluant strength is still 50%.
eluting agent is investigatedmeasure the NKA-9 type macroporous adsorbent resin dress post handled well in right amount, by optimal adsorption technological operation, after end upon adsorption, after a certain amount of washing, carry out eluting with 9BV 50% alcoholic solution, the every 1BV eluent of Fractional Collections, put 588nm place and measure effluent absorbance, calculate protein content and desorption efficiency, with 9 times of bed volumes for abscissa, in eluent, protein content is that vertical coordinate draws resin desorption curve, the results are shown in Table 5 and Fig. 3.Result shows, the increase desorbing along with eluant volume takes the lead in increasing rear minimizing, and when eluting agent reaches 5BV, desorption efficiency change is very little, and during 6BV, basic eluting is complete, therefore determines that eluting agent is 6BV.
demonstration testmeasure the NKA-9 type resin wet method handled well in right amount and be loaded on (blade diameter length ratio is 1:12) in 1.5cm × 30cm chromatographic column.Getting concentration is that 10mg/ml sample solution 25ml is with 2BVh
-1flow velocity continue through resin column.Collected post liquid, water lotion is collected after a certain amount of washing, eluting is carried out again with 5BV 50% ethanol, collect eluent, put the absorbance that 588nm place measured post liquid, water lotion and eluent, calculate resin absorption ration and desorption quantity, after the water-bath of resin elution liquid is volatilized, prepare sample by 2.1.3, measure PT, APTT and blood clotting-fibrinolytic Dynamic Graph, the results are shown in Table 6 and table 7.
Owing to have employed technique scheme, the technological progress that the present invention obtains is:
Reveal in the present invention in curve and can find out, applied sample amount when 40ml, slip increase severely, the applied sample amount therefore chosen at below 35ml, maximum possible decrease loss; The amount that the concentration of eluting solvent, consumption are selected after being all through a large amount of investigation, makes the desorption efficiency of object higher.
Accompanying drawing explanation
Fig. 1 is that Periostracum Cicadae water extracting alcohol hypostasis reveals curve;
Fig. 2 is that eluant strength is investigated;
Fig. 3 is that eluting agent is investigated.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention is described in further details:
Embodiment 1
Anticoagulant fibrinolysis component in a kind of Periostracum Cicadae, its isolation and purification method, is made up of following steps:
A, extraction: take Periostracum Cicadae fine medicinal material powder, add distilled water, reflux, extract, 3 times, each 1h, filters, merge extractive liquid, be concentrated into medicinal liquid than being 1:1, adding 95% ethanol to alcohol content is 90%, places sucking filtration after after spending the night, residue is dry, and obtain Periostracum Cicadae water extracting alcohol hypostasis, then adding distil water is mixed with concentration is 10mg/ml solution, ultrasonic 10min, centrifugal, get supernatant, obtain sample solution;
B, purification: take NKA-9 type resin, blade diameter length ratio is 1:12, and loading flow velocity is 2BVh
-1, loading volume is 25ml, collects post liquid, then carries out eluting with 50% ethanol of 5BV, and collect eluent, eluent water-bath volatilized, to obtain final product, recording desorption efficiency is 67.89%.
Embodiment 2
Anticoagulant fibrinolysis component in a kind of Periostracum Cicadae, its isolation and purification method, is made up of following steps:
A, extraction: take Periostracum Cicadae fine medicinal material powder, add distilled water, reflux, extract, 3 times, each 1h, filters, merge extractive liquid, be concentrated into medicinal liquid than being 1:1, adding 95% ethanol to alcohol content is 90%, places sucking filtration after after spending the night, residue is dry, and obtain Periostracum Cicadae water extracting alcohol hypostasis, then adding distil water is mixed with concentration is 10mg/ml solution, ultrasonic 10min, centrifugal, get supernatant, obtain sample solution;
B, purification: take NKA-9 type resin, blade diameter length ratio is 1:8, and loading flow velocity is 2BVh
-1, loading volume is 15ml, collects post liquid, then carries out eluting with 10% ethanol of 6BV, and collect eluent, eluent water-bath volatilized, to obtain final product, recording desorption efficiency is 66.78%.
Embodiment 3
Anticoagulant fibrinolysis component in a kind of Periostracum Cicadae, its isolation and purification method, is made up of following steps:
A, extraction: take Periostracum Cicadae fine medicinal material powder, add distilled water, reflux, extract, 3 times, each 1h, filters, merge extractive liquid, be concentrated into medicinal liquid than being 1:1, adding 95% ethanol to alcohol content is 90%, places sucking filtration after after spending the night, residue is dry, and obtain Periostracum Cicadae water extracting alcohol hypostasis, then adding distil water is mixed with concentration is 10mg/ml solution, ultrasonic 10min, centrifugal, get supernatant, obtain sample solution;
B, purification: take NKA-9 type resin, blade diameter length ratio is 1:10, and loading flow velocity is 2BVh
-1, loading volume is 35ml, collects post liquid, then carries out eluting with 40% ethanol of 5BV, and collect eluent, eluent water-bath volatilized, must record desorption efficiency is 65.79%.
Claims (6)
1. an anticoagulant fibrinolysis component in Periostracum Cicadae, is characterized in that in described Periostracum Cicadae, anticoagulant fibrinolysis component isolation and purification method is made up of following steps:
A, extraction: take Periostracum Cicadae fine medicinal material powder, add distilled water, reflux, extract, 3 times, each 1h, filters, merge extractive liquid, be concentrated into medicinal liquid than being 1:1, adding 95% ethanol to alcohol content is 90%, places sucking filtration after spending the night, residue is dry, and obtain Periostracum Cicadae water extracting alcohol hypostasis, then adding distil water is mixed with concentration is 10mg/ml solution, ultrasonic 10min, centrifugal, get supernatant, obtain sample solution;
B, purification: take macroporous adsorbent resin, blade diameter length ratio is 1:8-12, and loading flow velocity is 2BVh
-1, loading volume is 15-35ml, collects post liquid, then carries out eluting with the 10-50% ethanol of 5-6BV, collects eluent, eluent water-bath is volatilized, to obtain final product.
2. anticoagulant fibrinolysis component in Periostracum Cicadae according to claim 1, is characterized in that in described step B, blade diameter length ratio is 1:12.
3. anticoagulant fibrinolysis component in Periostracum Cicadae according to claim 1, is characterized in that in described step B, loading volume is 25ml.
4. anticoagulant fibrinolysis component in Periostracum Cicadae according to claim 1, is characterized in that in described step B, ethanol is 50%.
5. anticoagulant fibrinolysis component in Periostracum Cicadae according to claim 1, is characterized in that in described step B, macroporous adsorbent resin is NKA-9 type resin.
6. anticoagulant fibrinolysis component in Periostracum Cicadae according to claim 1, is characterized in that described method is made up of following steps:
A, extraction: take Periostracum Cicadae fine medicinal material powder, add distilled water, reflux, extract, 3 times, each 1h, filters, merge extractive liquid, be concentrated into medicinal liquid than being 1:1, adding 95% ethanol to alcohol content is 90%, places sucking filtration after after spending the night, residue is dry, and obtain Periostracum Cicadae water extracting alcohol hypostasis, then adding distil water is mixed with concentration is 10mg/ml solution, ultrasonic 10min, centrifugal, get supernatant, obtain sample solution;
B, purification: take NKA-9 type resin, blade diameter length ratio is 1:12, and loading flow velocity is 2BVh
-1, loading volume is 25ml, collects post liquid, then carries out eluting with 50% ethanol of 5BV, collects eluent, eluent water-bath is volatilized, to obtain final product.
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