CN101152211A - Dachengqi tang animal blood plasma freeze-dried powder and method for preparing the same - Google Patents
Dachengqi tang animal blood plasma freeze-dried powder and method for preparing the same Download PDFInfo
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- CN101152211A CN101152211A CNA2007100129934A CN200710012993A CN101152211A CN 101152211 A CN101152211 A CN 101152211A CN A2007100129934 A CNA2007100129934 A CN A2007100129934A CN 200710012993 A CN200710012993 A CN 200710012993A CN 101152211 A CN101152211 A CN 101152211A
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Abstract
The invention provides a potent purgative decoction animal plasma freeze-dried powder and the preparation method. The preparation method includes the following procedures: (1) rats are gavaged with the medical solution potent purgative decoction for three days; (2) 30 to 45 minutes after the last feeding, the rat blood is drawn by artery puncture, the heparin is anticoagulated, the plasma is isolated, and is divided evenly and stored in clean and aseptic vessels; (3) pre-frozen for 30 minutes under conditions of -20 degrees centigrade and frozen rapidly under conditions of -80 degrees centigrade; (4)the drug-containing plasma is frozen for 2 hours, frozen and dried to produce freeze-dried powder. The preparation method solves the equivalent adding problems such as the isolated storage of the effective components that interfere with the in vitro pharmacological experiment, further improves the consistency of in vitro traditional Chinese medicine pharmacological experiment and in vivo mechanism of practical drug action, and enhances the experimental reliability.
Description
Technical field
The invention belongs to the blood products technical field, relate in particular to the preparation method of pastille blood products.
Background technology
The Chinese medicine DACHENGQI TANG is that Tongli is captured method and represented prescription, and to peritonitis, gastrointestinal motility dysfunction etc. have significant curative effect.Previously the scholar carries out big quantity research to it, but or stay in the whole efficacy of medicine observing of experiment in the animal model, or be equal to the crude extract of DACHENGQI TANG that effective ingredient carries out experiment in vitro in the body, or infer the mechanism of action in its body with certain or the monomeric experiment in vitro of several effective ingredient.Because the correctly interior effect of antimer effective ingredient, so shortage experiment credibility reaches the concordance with the interior actual drug mechanism of action of body.Obviously lacking suitable research method has restricted DACHENGQI TANG drug effect and Its Mechanisms.And this type of medicine is often because of obtaining the admitted pharmacological evaluation report of energy and being refused by market, especially international market.
The serum pharmacological of Chinese medicine provides good idea for the research of the cellular/molecular level of Chinese Chinese medicine.The research method of serum pharmacological (Plasma Pharmacology) is after single medicinal material or Chinese traditional compound medicine are gavaged the animal certain hour, gather animal blood, separation of serum, carry out experiment in vitro with this pastille serum, disclosing a kind of experimental technique of mechanism of drug action by the biologic activity of research administration animal serum, is one of best approach of the external pharmacological experiment of present Chinese medicine.Replace decoct or crude extract to carry out experiment in vitro with pastille serum, got rid of of the interference of the physicochemical property of Chinese medicine crude preparation by using in the Chinese medicine experiment in vitro itself to experiment, can correctly reflect the medicine biotransformation in vivo and the pharmacodynamics effect of generation, have experiment condition controllability and reliable experiment result degree preferably.But not only reflect the direct effect of absorption portion in the medicine, and the indirect effect that can reflect homology material in the body that metabolite that ingredient forms and medicine induce under the body effect, help finding in the Chinese medicine compound real effective ingredient and effective site, for the further exploitation of medicine provides foundation.
Yet still there are some problems in the research method of serum pharmacological.The repetition that main is to actual drug mechanism in the body: after gavaging Chinese medicine to animal, at first entered blood by the Chinese medicine ingredients of gastrointestinal absorption.And serum is to remove fibrin and hemocyte behind the blood coagulation and the liquid that stays.In the blood coagulation process, except that a series of thrombins produce cascade reaction, also with activation in various degree or other variation of anticoagulation system, fibrinolytic system, anti-fibrinolytic system, complement system and kinin system, these processes may cause some effective ingredient or the Chinese medicine ingredients mediation endocrine bioactive substance of body (particularly protein and peptide class) to be degraded or loss of activity simultaneously.So serum can not be understood simply and become defibrinated blood plasma.Do not have serum in normal person and the most humans body, so pastille serum not necessarily reappears medicine at the intravital necessary being state of people.
In addition, the preservation of pastille serum also acquires a certain degree of difficulty, Zhou Mingmei etc. suppress platelet release 5-HT and block two of endotheliocyte calcium channels experimental studies have found that by carrying out pastille serum, serum is through (one 20 ℃ of long-time cryopreservation, 2 months) back drug effect significantly reduce (P＜0. O1), variations such as decomposition have taken place in active ingredient in the prompting preservation serum, cause content of drug effect components significantly to reduce.This with antibacterial experiment in pastille serum-20 ℃ preserve one week the back drug effect reduce about 7% and conform to, prompting pastille serum carries out cryogenic freezing to be preserved and still influences its drug effect, carries out the serum pharmacological experiment and is advisable to use short pastille serum of fresh serum or holding time.This has just limited the lasting progress of experiment and stability, homogeneity.
Therefore, be badly in need of a kind ofly being easy to preserve, easy to use and can reflect that DACHENGQI TANG enters the biological product of back time of day in the body.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of Wistar rat Dachengqi tang animal blood plasma freeze-dried powder.
The present invention takes medicine on the basis of Wistar rat pastille plasma drug level and pharmacokinetics process measuring DACHENGQI TANG, has set up the preparation method of following Wistar rat Dachengqi tang animal blood plasma freeze-dried powder.This method comprises the steps:
1. give Waist rats gavaged DACHENGQI TANG medicinal liquid, successive administration 3 days;
2. 30～45 minutes arterypunctures are got blood after the last administration, anticoagulant heparin, and separated plasma, equivalent is sub-packed in the cleaning sterile container then;
3.-20 behind ℃ pre-freeze 30min ,-80 ℃ of quick freezing are solid;
4. after pastille blood plasma freezes solid 2h, make lyophilized powder through lyophilization.
Wherein, described step concrete operation method 1. is: will hold the aerated particle electuary greatly and be formulated as 100% equivalent medicinal liquid, and press the dosage of 1ml/ (100g body weight) and give Waist rats gavaged, every day twice, successive administration 3 days.
No matter the preparation method of above-mentioned Dachengqi tang animal blood plasma freeze-dried powder in which kind of concrete mode operates, after the preferred last administration of the blood time of getting 2. of described step 45 minutes.
More preferably, 2. the step of the preparation method of above-mentioned Dachengqi tang animal blood plasma freeze-dried powder can carry out according to following concrete operations scheme: after the last administration under 45 minutes chloral hydrate 3ml/Kg intraperitoneal injection of anesthesia, femoral artery puncture is got blood, anticoagulant heparin, separated plasma, equivalent is sub-packed in the cleaning sterile container then.
More specifically, the preparation method of Dachengqi tang animal blood plasma freeze-dried powder of the present invention specifically comprises the steps:
1. will hold the aerated particle electuary greatly and be formulated as 100% equivalent medicinal liquid, and press 1ml/100g dosage and give Waist rats gavaged, every day twice, successive administration 3 days;
2. after the last administration under 45 minutes chloral hydrate 3ml/Kg intraperitoneal injection of anesthesia, femoral artery puncture is got blood, anticoagulant heparin, and separated plasma, equivalent is sub-packed in the EP of cleaning sterile then;
3.-20 behind ℃ pre-freeze 30min ,-80 ℃ of quick freezing are solid;
4. the pastille plasma sample is used to prepare lyophilized powder after freezing solid 2h: start freezer dryer, and vacuum pump preheating 30min, chilling temperature drops to minimum; To freeze solid plasma sample and put into plug-in bottle, last machine begins main lyophilizing work under-50 ℃; Close after the valve between the lyophilizing cavity condenser, the intravital pressure in lyophilizing chamber does not significantly raise, and then can finish lyophilizing; Close vacuum pump, charge into aseptic drying nitrogen, take out the blood plasma lyophilized powder and preserve in-20 ℃ of close dryings.
Content of the present invention also comprises the Dachengqi tang animal blood plasma freeze-dried powder for preparing by any one method in the said method.
Dachengqi tang animal blood plasma freeze-dried powder of the present invention and preparation method thereof is based on takes medicine on the basis of Wistar rat pastille plasma drug level and pharmacokinetics process data collection analysis to DACHENGQI TANG.
At first need to determine dosage regimen and blood sampling time, the concrete grammar that is adopted among the present invention is: (300 ± 20g) 50 is at interval with 15min, is divided into eight groups, 5 every group to get the Wistar rat.Gavage and hold gas electuary solution greatly, every day twice, successive administration 3 days.Each treated animal is respectively at 15min, 30min, 45min, 60min, 75min, 90min, 105min, 120min arterial blood extracting, separated plasma after the last administration.Detect emodin, magnolol concentration in the blood plasma with RP-HPLC, draw plasma concentration curve, find out blood medicine peak concentration time, as the plasma sample acquisition time of taking medicine.
The present invention has also detected the influence of the Dachengqi tang animal blood plasma freeze-dried powder pair cell that makes by said method and the variation of active constituent content after long preservation.
The preparation method of DACHENGQI TANG pastille blood plasma lyophilized powder of the present invention is selected the Wistar rat of extensive utilization for use, easy acquisition is arranged, raise easily, get blood convenient, with the proximate advantage of human biology's characteristic.Replace pastille serum with pastille blood plasma, more approaching middle pharmaceutically active ingredient true process in vivo.Avoid the loss of the effective ingredient that coagulation process causes, got rid of coagulation process and may cause in some pharmaceutically active ingredient or Chinese medicine ingredients mediation body endocrine bioactive substance (particularly protein and peptide class) to be degraded and reduce or lose drug effect; Also avoid simultaneously coagulation process with the activation of systems such as fibrinolytic, complement, kassinin kinin and stimulated by thrombin platelet and of the interference of leukocyte release large number of biological active substance to experimental result.Adopt Freeze Drying Technique to prepare pastille blood plasma lyophilized powder, get rid of the influence of factors such as temperature, air oxidation, self metabolism decomposition, can preserve in the pastille blood plasma effective ingredient to greatest extent and activate the endogenous bioactive substance that body produces; And the interior blood drug level of drug level consubstantiality in the vitro reactions system is equated, solved the difficult problem that separation is preserved and equivalence is added that perplexs effective ingredient in the external pharmacological evaluation, further improve the concordance and the experiment credibility of actual drug mechanism of action in external herbal pharmacology experiment and the body, realized stabilisation, the serialization of external herbal pharmacology experiment.
Description of drawings
Accompanying drawing 6 width of cloth of the present invention:
Fig. 1 is the HPLC collection of illustrative plates of emodin, magnolol hybrid standard product;
Fig. 2 is the HPLC collection of illustrative plates of pastille plasma sample;
Emodin content in Fig. 3 Wistar rat plasma-blood specimen collection time graph;
Magnolol content-blood specimen collection time graph in Fig. 4 Wistar rat plasma;
Fig. 5 is the relative amount curve of emodin in the pastille plasma sample of preserving with lyophilized powder and freezing preservation form respectively;
Fig. 6 is the relative amount curve of magnolol in the pastille plasma sample of preserving with lyophilized powder and freezing preservation form respectively.
Wherein: among Fig. 5 :-■-be emodin relative amount in the pastille blood plasma lyophilized powder; Emodin relative amount in the pastille blood plasma of--be freezing; Among Fig. 6 :-■-be magnolol relative amount in the pastille blood plasma lyophilized powder; Magnolol relative amount in the pastille blood plasma of--be freezing.
The specific embodiment
Present embodiment is further detailed the preparation method of Dachengqi tang animal blood plasma freeze-dried powder of the present invention, and this embodiment is limited content of the present invention never in any form.
1. detection method
If no special instructions, to active component in the pastille plasma sample, the content detection of emodin, magnolol all adopts the RP-HPLC method among the present invention, and testing conditions is:
Checkout equipment: Waters high performance liquid chromatograph
Chromatographic column: Nava-pak C
1860A, 4,3.9 * 150mm
Detect wavelength: detect wavelength 294nm
Column temperature: 30 ℃
Sample size: 20 μ l
Flow velocity: flow velocity 1ml/min
Mobile phase: acetonitrile: water (0.125% acetic acid, 0.4% sodium acetate) 60: 40
The t of emodin, magnolol under this condition
RBe respectively 3.829min and 4.861min, the two and biological impurity separate good.The HPLC separating resulting of emodin, magnolol hybrid standard product and pastille plasma sample is respectively shown in accompanying drawing 1, accompanying drawing 2.
2. dosage regimen and blood sampling time are determined
Get Wistar rat (300 ± 20g) 50 are divided into eight groups, 5 every group.That gets that Tianjin hospital of Nankai produces holds aerated particle electuary commodity greatly, is formulated as 100% equivalent crude drug liquid, gavages the Wistar rat by 1ml/100g dosage, every day twice, successive administration 3 days.Each treated animal is got blood respectively at femoral artery puncture under 15min, 30min, 45min, 60min, 75min, 90min, 105min, the 120min chloral hydrate 3ml/Kg intraperitoneal injection of anesthesia after the last administration, anticoagulant heparin, 4 ℃, the centrifugal 10min of 4000r/min, separated plasma.
Get blood plasma 0.5ml, add 0.5ml ether vortex vibration mixing 1min, the centrifugal 10min of 12000r/min gets organic layer, and continuous extraction three times merges organic facies, and 40 ℃ of water-baths volatilize, the dissolving of 0.06ml acetonitrile sonic oscillation, 20 μ l sample introductions.Detect emodin, magnolol concentration in the blood plasma with RP-HPLC, draw plasma concentration curve, find out blood medicine peak concentration time, as the plasma sample acquisition time of taking medicine.
To the analysis result of emodin honokiol respectively shown in accompanying drawing 3 and accompanying drawing 4:
The blood drug level of each time point is analyzed (P＜0.01) through the SPSS13 statistical software in the accompanying drawing 3: each time point blood drug level has marked difference, and emodin concentration peak 45min after the last administration obtains in the blood plasma.
The blood drug level of each time point is compared (P＜0.01) through analysis (P＜0.01): 30min of SPSS13 statistical software and 45min group with other each time point blood drug level in the accompanying drawing 4, and marked difference is arranged; 30min and 45min group (P＞0.05) indifference, the blood specimen collection time can be after the last administration 30～45min.
Comprehensive The above results, the sampled plasma time is determined at 45min after the last administration.
3. the preparation of the collection of pastille plasma sample, processing and Dachengqi tang animal blood plasma freeze-dried powder
That gets that Tianjin hospital of Nankai produces holds aerated particle electuary commodity greatly, is formulated as 100% equivalent crude drug liquid, gavages the Wistar rat by 1ml/100g dosage, every day twice, successive administration 3 days.Last administration 45min is after femoral artery is got blood, anticoagulant heparin, 4 ℃, the centrifugal 10min separated plasma of 4000r/min.Equivalent is sub-packed in clean aseptic EP pipe or the test tube.-80 ℃ of quick freezing are solid behind-20 ℃ of pre-freeze 30min.
The pastille plasma sample promptly can be used for preparing lyophilized powder after freezing solid 2h.Start instrument, vacuum pump preheating 30min, condenser temperature drops to minimum.To freeze solid sample and put into plug-in bottle, last machine begins main lyophilizing work 12h under-50 ℃.Close after the valve between the lyophilizing cavity condenser, the intravital pressure in lyophilizing chamber does not significantly raise, and illustrates that then drying finishes substantially, can finish lyophilizing.Close vacuum pump, charge into aseptic drying nitrogen, take out in-20 ℃ of close dryings and preserve.
4.MTT detect the influence of different pastille blood plasma addition pair cells
MTT detects the influence of different pastille blood plasma addition on cell proliferation: it is good to get growth conditions, is in the Waist rat small intestine ICC cell in 2～3 generations of exponential phase, adds an amount of 0.25% trypsinization.The blood cell counting plate counting is made into 1 * 10 with DMEM/F-12 whole part culture fluid (containing 20% hyclone)
5Individual/the ml single cell suspension.With 1 * 10
4Individual/hole density is inoculated in 96 well culture plates, every pore volume 0.2ml.The hole of plate periphery does not add cell, prevents edge effect.The hole string zeroing that blanks does not add cell and makes blank only to add culture fluid.Shake up with the dull and stereotyped shaking table 5min slow-speed of revolution and to make the cell uniform distribution.At 37 ℃, 5% CO
2Incubator in behind the adhere-wall culture 1h, change and contain 5% hyclone DMEM/F-12 whole part culture fluid and cultivate 24h, make cell synchronization.Serum-free DMEM/F-12 culture medium equal-volume dissolving Waist rat is held the gas blood plasma lyophilized powder of taking medicine greatly, if take medicine blood plasma cultivate 0.5 hour, 1 hour, 24 hours three groups, every group of 5%, 10%, 20%, 50%, 75%, 100% pastille plasma concentration of all preparing with the frozen dry blood plasma powder adds, every group three multiple hole, every hole 0.2ml.Negative control group adds no hyclone DMEM/F-12 culture medium.Abandon culture supernatant in the hole respectively at cultivating after 0.5 hour, 1 hour, 24 hours, serum-free DMEM/F-12 culture medium preparation 5mg/mlMTT solution, every hole adds 0.02ml, and 37 ℃ are continued to hatch and made MTT be reduced to Formazan in 4 hours.Stop cultivating, abandon supernatant in the hole.Every hole adds 0.15mlDMSO, vibrates 10 minutes, and Formazan is fully dissolved, and under the 570nm wavelength, measures each hole absorbance value (OD) with enzyme-linked immunosorbent assay instrument, obtains cell survival rate by following formula, promptly
Cell survival rate=(experimental group OD value-blank group OD value)/(negative control group OD value-blank group OD value) * 100%.
The result is as shown in the table:
| Incubation time h | Plasma drug level (v/v) | |||||
| 100％ | 75％ | 50％ | 20％ | 10％ | 5％ | |
| 0.5 1 24 | 1.54 1.45 1.35 | 1.53 1.47 1.43 | 1.54 1.55 1.56 | 1.56 1.57 1.60 | 1.45 1.50 1.58 | 1.39 1.43 1.46 |
| Blank | 0.952 | 0.968 | 0.983 | 0.992 | 1.101 | 1.109 |
| Negative | 1.380 | 1.498 | 1.343 | 1.468 | 1.431 | 1.432 |
According to The above results, pastille blood plasma lyophilized powder has satisfied pharmacologically active.
Long preservation to the influence of main effective ingredient (emodin, magnolol) content in the Dachengqi tang animal blood plasma freeze-dried powder and with the comparison of freezing store method.
Getting equivalent pastille blood plasma preserves with lyophilized powder and freezing mode respectively, regularly change with active constituent content in the RP-HPLC monitoring pastille blood plasma, with the 0th day content was that 100% active constituent content that calculates sampling time point accounts for the percentage ratio of preserving content when initial, and with this curve plotting, its result as shown in Figure 5 and Figure 6.
According to Fig. 5 as seen, preserve DACHENGQI TANG pastille blood plasma with the form of lyophilized powder of the present invention, in a long time, the content of active ingredient emodin almost remains unchanged in the sample; In comparison, emodin content prolongs with the holding time and is downward trend by a relatively large margin in the pastille blood plasma of preserving in the freezing mode, so lyophilized powder is preserved and preserved more effective than freezing.
Equally, according to Fig. 6 as seen, preserve DACHENGQI TANG pastille blood plasma with the form of lyophilized powder of the present invention, in a long time, the content of effective ingredient magnolol almost remains unchanged in the sample; In comparison, the content of magnolol prolongs with the holding time and is downward trend by a relatively large margin in the pastille blood plasma of preserving in the freezing mode, so lyophilized powder is preserved and preserved more effective than freezing.
Claims (6)
1. the preparation method of Dachengqi tang animal blood plasma freeze-dried powder is characterized in that it comprises the steps:
1. give Waist rats gavaged DACHENGQI TANG medicinal liquid, successive administration 3 days;
2. 30～45 minutes arterypunctures are got blood after the last administration, anticoagulant heparin, and separated plasma, equivalent is sub-packed in the cleaning sterile container then;
3.-20 behind ℃ pre-freeze 30min ,-80 ℃ of quick freezing are solid;
4. after pastille blood plasma freezes solid 2h, make lyophilized powder through lyophilization.
2. the preparation method of Dachengqi tang animal blood plasma freeze-dried powder according to claim 1, it is characterized in that described step concrete operation method 1. is: will hold the aerated particle electuary greatly and be formulated as 100% equivalent medicinal liquid, press the dosage of 1ml/ (100g body weight) and give the Waist rats gavaged, every day twice, successive administration 3 days.
3. the preparation method of Dachengqi tang animal blood plasma freeze-dried powder according to claim 1 and 2 is characterized in that the described step blood time of getting 2. is after the last administration 45 minutes.
4. the preparation method of Dachengqi tang animal blood plasma freeze-dried powder according to claim 1 and 2, it is characterized in that described step 2. concrete operation method be: after the last administration under 45 minutes chloral hydrate 3ml/Kg intraperitoneal injection of anesthesia, femoral artery puncture is got blood, anticoagulant heparin, separated plasma, equivalent is sub-packed in the cleaning sterile container then.
5. the preparation method of Dachengqi tang animal blood plasma freeze-dried powder according to claim 1 is characterized in that the concrete steps of this method comprise:
1. will hold the aerated particle electuary greatly and be formulated as 100% equivalent medicinal liquid, and press 1ml/100g dosage and give Waist rats gavaged, every day twice, successive administration 3 days;
2. after the last administration under 45 minutes chloral hydrate 3ml/Kg intraperitoneal injection of anesthesia, femoral artery puncture is got blood, anticoagulant heparin, and separated plasma, equivalent is sub-packed in the EP of cleaning sterile then;
3.-20 behind ℃ pre-freeze 30min ,-80 ℃ of quick freezing are solid;
4. the pastille plasma sample is used to prepare lyophilized powder after freezing solid 2h: start freezer dryer, and vacuum pump preheating 30min, chilling temperature drops to minimum; To freeze solid plasma sample and put into plug-in bottle, last machine begins main lyophilizing work under-50 ℃; Close after the valve between the lyophilizing cavity condenser, the intravital pressure in lyophilizing chamber does not significantly raise, and then can finish lyophilizing; Close vacuum pump, charge into aseptic drying nitrogen, take out the blood plasma lyophilized powder and preserve in-20 ℃ of close dryings.
6. pass through the Dachengqi tang animal blood plasma freeze-dried powder of claim 1,2,3 or 5 described methods preparations.
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- 2007-09-28 CN CNA2007100129934A patent/CN101152211A/en active Pending
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| US11604026B2 (en) | 2019-03-14 | 2023-03-14 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
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| US11740019B2 (en) | 2019-03-14 | 2023-08-29 | Terumo Bct Biotechnologies, Llc | Lyophilization loading tray assembly and system |
| US11747082B2 (en) | 2019-03-14 | 2023-09-05 | Terumo Bct Biotechnologies, Llc | Multi-part lyophilization container and method of use |
| US11815311B2 (en) | 2019-03-14 | 2023-11-14 | Terumo Bct Biotechnologies, Llc | Lyophilization container fill fixture, system and method of use |
| CN110243965A (en) * | 2019-06-25 | 2019-09-17 | 四川大学华西医院 | A kind of detection method of tiger fur dachengqi decoction |
| CN110243965B (en) * | 2019-06-25 | 2021-09-03 | 四川大学华西医院 | Detection method of Tiger skin Dachengqi soup |
| CN111077242A (en) * | 2019-12-05 | 2020-04-28 | 广西国际壮医医院 | High performance liquid chromatography analysis method of traditional Chinese medicine formula Dachengqi decoction |
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