CN1785261A - Hotseason grow corydaline, its preparation method and its medicinal composition - Google Patents
Hotseason grow corydaline, its preparation method and its medicinal composition Download PDFInfo
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- CN1785261A CN1785261A CN 200510012978 CN200510012978A CN1785261A CN 1785261 A CN1785261 A CN 1785261A CN 200510012978 CN200510012978 CN 200510012978 CN 200510012978 A CN200510012978 A CN 200510012978A CN 1785261 A CN1785261 A CN 1785261A
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Abstract
A corydaline for preparing medicines to prevent and treat acute cerebral sichemia is prepared from the drived tuber of corydalis, and contains protopine, bicuculline, tetraphdropalmatine, palmatine, etc. Its preparing process and its composite medicine are also disclosed.
Description
Technical field
The present invention relates to a kind of Hotseason grow corydaline and preparation method thereof and its pharmaceutical composition, and the application in preparation treatment and prophylaxis of acute brain ischemia medicament.
Background technology
The dry tuber of bloodroot Hotseason grow Corydalis decumbens (Thunb) pers is promoting flow of QI and blood, a removing obstruction in the collateral to relieve pain medicine commonly used, Chinese medicine name Rhizoma Corydalis Decumbentis.Can be used for many disturbance of blood circulation treatment of diseases such as apoplectic hemiplegia, traumatic injury, rheumatic arthritis, sciatica.At present both at home and abroad existing Rhizoma Corydalis Decumbentis tablet and Rhizoma corydalis decumbenti Injection production listing, Rhizoma Corydalis Decumbentis tablet be by the crude drug Rhizoma Corydalis Decumbentis beat that powder mixes with its alcohol-extracted extract, granulation, tabletting form; Its injection then be by the crude drug Rhizoma Corydalis Decumbentis through aqueous extraction-alcohol precipitation technology, according to a conventional method through fill, step such as seal and make, but can not intravenously administrable.These preparations, technology is coarse, and inclusions is indeterminate, and inherent quality is difficult to control, does not therefore meet the trend of modern Chinese medicine preparation development.
Nineteen eighty-two " Jiangxi Province's drug standard " at first uses dilute hydrochloric acid to extract with percolation, collect filtrate then and carry out ion exchange by cation exchange resin, behind alcoholic solution (PH9-10) the gradation backflow eluting with ammonia, collect eluent and promptly obtain the Rhizoma Corydalis Decumbentis alkaloid extract.The shortcoming of this method is wherein to have used to human body and the deleterious reagent of environment, and the alkaloidal response rate is lower.
The composition of Chinese patent 02111103.0 described total alkaloid of corydalis rhizome is: multiple alkaloid such as summer alkali-free, bicuculline, jateorhizine, a-tetrahydrochysene palm leaf coculine and protopine etc.The purity of said total alkaloid of corydalis rhizome is more than 80%.But wherein mainly be protopine, and its content account for more than 50% of total alkaloids.This patent does not have the ratio of other components and offers some clarification on.Limiting the quantity of of hypertoxicity composition bicuculline do not have clearly regulation, queries so its safety exists.Extracting method: after taking by weighing and cleaning decumbent corydalis as medicine material, add dilute hydrochloric acid (0.1-1%) aqueous solution soaking excessive more than 10 times and boiled 0.5-2 hour.After the simple filtration, collect medicinal residues and, continued to boil 0.5-2 hour to the dilute hydrochloric acid that wherein adds 5-10 times of volume (0.05-5.5%) aqueous solution.Behind the conventional filtration, collect filtrate and above-mentioned filtrate is merged.With sodium hydroxide solution the pH value of filtrate is transferred to 7, crosses macroporous adsorptive resins (flow velocity 5ml/ branch) then.When monitoring stream fluid alkaloid is positive according to a conventional method absorption saturated, be washed to colourless after, concentration (30-60%) and high concentration (60-90%) ethanol elution in using successively.After monitoring bio alkali content is negative and reacts, collect also merging eluent.Then eluent peroxidating aluminium lamination sheet-pile (flow velocity 5ml/ branch) is removed impurity with absorption.Behind the decompression recycling ethanol, required product and the conventional filtration of stripping under acid condition (PH1-5).Use sodium hydroxide solution that the pH value of eluate is transferred to 8-11 then, to separate out precipitation.Promptly obtain being used for Rhizoma corydalis decumbentis total alkaloids of the present invention after merging precipitate.
The composition of Chinese patent 03118261.5 described total alkaloid of corydalis rhizome is: multiple alkaloids such as protopine, bicuculline, jateorhizine, a-tetrahydropalmatine and palmatine.The purity of said total alkaloid of corydalis rhizome is more than 80%.The ratio of total alkaloids component is clear and definite: protopine: 25-65%, tetrahydropalmatine: 10-25%, Bicuculline: 5-10%, fibrauretin: 10-25%, other biological alkali and extract: 5-20%.Limiting the quantity of of hypertoxicity composition bicuculline is 5-10%.Extracting method: with the raw material of doing nothing summer, add an amount of polar solvent percolation and extract, collect percolate to the inanimate object alkali reaction, merging filtrate, concentrate and obtain concentrated solution, the concentrated solution that obtains through above-mentioned steps is carried out the absorption with macroporous adsorbent resin chromatography, remove impurity with the aqueous alkali eluting; Or,, collect eluent again with polarity eluent eluting total alkaloid of corydalis rhizome with resin absorption impurity, and reclaim solvent, be concentrated in the alcoholic solution crystallization promptly.
Modern pharmacology studies show that Hotseason grow contains multiple alkaloid with remarkable physiologically active, but wherein a kind of alkaloid toxicity of Bicuculline by name is bigger, the rat vein administration, and median lethal dose(LD 50) is 0.85mg/Kg; And content accounts for more than 15% of total alkaloids in the medical material.Should remove when being extraction separation, content is low more in the preparation, and medication is then safe more.
Above several method extraction separation gained total alkaloids.Because of the technology difference, so inherent component also has very big difference, which kind of extracting method all exists toxic component Bicuculline content higher, and can not quantitative shortcoming, although therefore make that the total alkaloid of corydalis rhizome drug effect is remarkable, because there is safety hidden danger in it, clinical practice is restricted.
Summary of the invention
The technical problem to be solved in the present invention is, at above-mentioned the deficiencies in the prior art, component in the Rhizoma Corydalis Decumbentis is adopted special extraction process, hypertoxicity composition Bicuculline is reduced to below 5%, several alkaloidal compositions are enriched to purity more than 80%, and set up the preparation method of extracting described total alkaloids, preparation method and the application in preparation treatment and prophylaxis of acute brain ischemia medicament thereof that this total alkaloids is made the freeze-dried powder that can supply intravenously administrable are proposed.
The described Hotseason grow corydaline of this patent, by Rhizoma Corydalis Decumbentis is the dry tuber extraction of bloodroot Hotseason grow Corydalisdecumbens (Thunb) pers, by weight percentage, total alkali wherein contains protopine (protopine) 35-70%, Bicuculline (bicuculline)≤5%, tetrahydropalmatine (tetrahydropalmatine), fibrauretin (palmatine), papaverine, dihydro Bicuculline, bulbocapnine, bulbocapnine etc. more than 80%.
For effectively controlling Hotseason grow corydaline, protopine, Bicuculline content, the present invention sets up following content assaying method.
Adopt the content of its protopine of high effective liquid chromatography for measuring:
Chromatographic column: octadecylsilane chemically bonded silica is filler AichromBond-AQ C
185 m (4.6 * 150mm) chromatographic columns (U.S.)
Detect wavelength: 290nm, flow velocity: 1.0ml/min, column temperature: 30 ℃
Mobile phase: acetonitrile-0.1% trifluoracetic acid (24: 76)
Accurate reference substance solution and each 20 l of need testing solution of drawing inject high performance liquid chromatograph during mensuration, measure the content that promptly gets protopine.
Adopt the content of its Bicuculline of high effective liquid chromatography for measuring:
Instrument and equipment: high performance liquid chromatograph LC-10ATvp pump, SPD-10Avp detector (day island proper Tianjin company); Ultraviolet spectrophotometer UV-1700 (day island proper Tianjin company); 2010 chromatographic work stations (intelligent information engineering research institute of Zhejiang University); Ultrasonic cleaner CQ-10 (Guangzhou jewel Electrical Appliances Co., Ltd)
Assay method
Chromatographic column: octadecylsilane chemically bonded silica is filler AichromBond-AQ C
185 m (4.6 * 150mm) chromatographic columns (U.S.)
Detect wavelength: 225nm, flow velocity: 1.0ml/min, column temperature: 30 ℃
Mobile phase: acetonitrile-0.1% trifluoracetic acid (24: 76)
Accurate reference substance solution and each 20 l of need testing solution of drawing inject high performance liquid chromatograph during mensuration, measure the content that promptly gets Bicuculline.
Adopt the UV method to measure total alkaloid contents:
Instrument and equipment: ultraviolet spectrophotometer UV-1700 (day island proper Tianjin company); Ultrasonic cleaner CQ-10 (Guangzhou jewel Electrical Appliances Co., Ltd)
Accurate absorption need testing solution is an amount of during mensuration, operate by the method under the standard curve preparation in accordance with the law, test according to an appendix VB of Chinese Pharmacopoeia version in 2000 spectrophotography, in 415nm wavelength place scanning (is blank with corresponding solution), measure the absorption maximum degree, read the amount of total alkaloids from corresponding standard curve.
The preparation method of Hotseason grow corydaline:
(1) be raw material with the Hotseason grow tuber, the polar solvent percolation that adds 2-20 times extracts, or the forced refluence extraction, collects extracting solution to the inanimate object alkali reaction, and merge extractive liquid, is transferred PH to 6-7 with alkali liquor, is concentrated in right amount, obtains extracting concentrated solution;
(2) the extraction concentrated solution that above-mentioned steps is obtained carries out the macroporous resin adsorption chromatography, and with the 1-8mol/l strong base solution washing of temperature more than 40 ℃, consumption is 1-10 a times of column volume, again washing roguing of the alcoholic solution of 50-60% and hypertoxicity composition Bicuculline.
(3) with polarity eluent eluting Hotseason grow alkaloid, collect eluent, reclaim solvent, be concentrated into small size.
(4) transfer pH value to alkalescence with alkali liquor, separate out precipitation, the leaching precipitation, saturated common salt is washed to neutrality, drying.
(5) the precipitation organic solvent dissolution filters, and filtrate is reclaimed solvent to small size, places crystallize, and the leaching crystallizing and drying promptly gets Hotseason grow corydaline.
Wherein used polar solvent is selected from phosphoric acid, hydrochloric acid, citric acid, tartaric acid, the succinic acid solution of 1-10% or the ethanol of 75-95% or methanol solution in the step (1); Better polar solvent is selected the phosphoric acid of 2-3% for use;
Wherein big pore adsorption resin is D in the step (2)
101A, DM
201, DM
301, D
301In a kind of.
Wherein the used polarity eluent sour water that is used for eluting Hotseason grow alkali is that hydrochloric acid, phosphoric acid, lactic acid, succinic acid solution or the concentration of 0.1-10% is 60-95% acidic ethanol or acidic methanol solution in the step (3).
Wherein used alkali liquor is that concentration is NaOH or the KOH solution of 5-20% in the step (4).
Wherein in the step (5) used organic solvent be chloroform or≤C
4Lower aliphatic alcohols, acetone, ethyl acetate.
The pharmaceutical composition that contains Hotseason grow corydaline:
Adopt this method to extract the Hotseason grow corydaline that obtains, dose conventional pharmaceutical excipient, promptly can be made into oral, injection type.
A kind of preparation method of injection Hotseason grow corydaline freeze-dried powder is:
1) the Hotseason grow corydaline injection prescription of freeze-drying powder is formed:
Hotseason grow corydaline 1～5g, add 0.5～10% (percentage by weight) acid solution, 5～10ml (being selected from hydrochloric acid, phosphoric acid, lactic acid or succinic acid), excipient 20～5000g (be selected from mannitol, dextran, glucose or the sodium chloride one or both or two or more), add the injection water make 1000ml in right amount.
Optimum formula is: Hotseason grow corydaline 2g, and 1% acid (phosphoric acid) solution 10ml, mannitol 50g adds the injection water and makes 1000ml in right amount.
2) preparation technology
Get the Hotseason grow corydaline extract, dissolve with acid solution, it is an amount of to add the injection water, stirring makes abundant dissolving, other gets mannitol and adds in an amount of water for injection after the stirring and dissolving, merge two solution, be mixed and transfer PH to 3～5 with 1%NaOH solution, add an amount of water for injection again and be settled to 1000ml, add an amount of needle-use activated carbon stirring and boil 30min, the microporous filter membrane classification filters, and the filtrate quantitative aseptic is sub-packed in the good control antibiotic glass bottle of aseptic process, lid is jumped a queue, rolled to vacuum lyophilization promptly.Every bottle of specification is 1mg or 2mg.
The present invention also proposes the application of a kind of Hotseason grow corydaline in preparation treatment and prevention ischemic cerebrovascular medicine.
1, the injection Hotseason grow corydaline is to the influence of focal cerebral ischemia in rats
Test causes the focal cerebral ischemia model with electrocoagulation blocking-up rat brain medium-sized artery, and intravenously administrable is observed the therapeutical effect of injection Hotseason grow corydaline to focal cerebral ischemia.The result shows, compares with model group, and injection Hotseason grow corydaline group rat focus of infarct obviously dwindles, the neurobehavioral obstacle of animal obviously alleviates behind the cerebral infarction, activity of SOD in serum obviously strengthens, and pathological examination shows that administration group rat brain edema obviously alleviates, and nervous tissue pathological change obviously improves.Illustrate that the injection Hotseason grow corydaline has therapeutical effect to focal cerebral ischemia in rats.
2, the injection Hotseason grow corydaline is to the influence of rat whole brain ischemia
Test merges the method that blood-letting brings high blood pressure down with the ligation bilateral common carotid arteries, causes the rat whole brain ischemia model, observes the influence of injection Hotseason grow corydaline to rat brain index, brain water content.The result shows that behind this medicine of intravenous injection, rat brain index, brain water content obviously reduce, with the remarkable (p＜0.05-0.01) of model group comparing difference.Illustrate that the injection Hotseason grow corydaline has protective effect to rat cerebral ischemia.
3, the injection Hotseason grow corydaline is to the influence of anesthetized dog cerebral blood flow
This test is main observation index with the ICAF amount, observes the influence of injection Hotseason grow corydaline to the animal brain blood flow.The result shows that the injection Hotseason grow corydaline can significantly increase the dog cerebral blood flow, reduces cerebral vascular resistance, and arteriotony, heart rate, electrocardiogram are not had obvious influence.
4, the injection Hotseason grow corydaline is to the influence of chmice acute cerebral ischemia
Test causes the chmice acute cerebral ischemic model with the method for forfeiting, and observes the effect of medicine.Result of the test shows that after vein gave the injection Hotseason grow corydaline, the mouse breathing time obviously prolonged.Point out this medicine that acute cerebral ischemia is had protective effect.
5, the injection Hotseason grow corydaline is to the microcirculatory influence of mice pia mater encephali
Test causes the rat's pial microcirculation disturbance by injecting 15% high molecular dextran, observes the influence of injection Hotseason grow corydaline to the rat microcirculation disturbance.The result shows, 0.4mg/kg, 0.2mg/kg group all can obviously increase velocity of blood flow at observed 10,20,30min time point behind the injection Hotseason grow corydaline intravenous administration, improve the blood fluidised form, 0.1mg/kg group presents certain dose-effect relationship in 30min have clear improvement velocity of blood flow and fluidised form effect.Prompting injection Hotseason grow corydaline has the effect that improves the rat's pial microcirculation disturbance.
6, the injection Hotseason grow corydaline is to the influence of rabbit platelet aggregation
The BornShi turbidimetry is adopted in test, observes the influence of injection Hotseason grow corydaline to the rabbit platelet aggregation.The result shows: give rabbit auricular vein drug administration by injection for three days on end, the injection Hotseason grow corydaline obviously reduces adenosine diphosphate (ADP) (ADP) and collagen-induced rabbit platelet aggregation rate (P＜0.05～0.01), and the inductive rabbit platelet aggregation rate of arachidonic acid (AA) is had reduction trend.Prompting injection Hotseason grow corydaline has the effect of anticoagulant.
7, the injection Hotseason grow corydaline is to the influence of rats in vitro thrombosis and blood viscosity
Experimental observation injection Hotseason grow corydaline is to the influence of rats in vitro thrombosis and blood viscosity.The result shows: rat tail vein injection administration for three days on end, injection Hotseason grow corydaline can obviously be shortened thrombosis length (P＜0.05～0.01), obviously alleviate wet weight of thrombus and dry weight (P＜0.01～0.001); Obviously reduce shear rate 30S
-1And 5S
-1Under whole blood viscosity (P＜0.05).Prompting injection Hotseason grow corydaline has the effect that suppresses thrombosis, blood viscosity lowering.
The present invention takes 1-10% acid extraction or 75-95% ethanol, methanol extraction, and absorption with macroporous adsorbent resin after about 5% alkali liquor eluting is removed Bicuculline and impurity more than 40 ℃, makes the higher Hotseason grow corydaline of a purity.They can remove impurity such as aminoacid and saccharide, tannin effectively, particularly reduce the content of toxic component Bicuculline, obviously improved the purity of Hotseason grow corydaline, the Hotseason grow total alkaloids that method of the present invention prepares gained does not all have aminoacid and free sugar and tannin reaction, adopts UV method and HPLC method to record its content 〉=80%; The speckle of relevant position clear-cuts such as all visible and former protopine of reference substance of its thin-layer chromatogram, tetrahydropalmatine, fibrauretin, papaverine, dihydro Bicuculline, bulbocapnine, bicuculline≤5%, point out Hotseason grow corydaline purity height of the present invention, the severe toxicity component content is low, medication is safer, uses more extensively, and this method is easy and simple to handle, being easy to grasp, is effective, feasible Hotseason grow corydaline preparation method.
Description of drawings
Fig. 1 and Fig. 2 are the fingerprint images that shows that the present invention (embodiment 1) total alkaloids active ingredient exists, and wherein Fig. 1 is the Hotseason grow corydaline thin layer chromatography, and Fig. 2 is a high performance liquid chromatogram HPLC fingerprint image.
Among Fig. 1: 1-protopine, 2-Bicuculline, 3-tetrahydropalmatine, 4-Hotseason grow corydaline, 5-fibrauretin, 6-dihydro Bicuculline, 7-bulbocapnine.
Thin layer chromatography condition: adsorbent: silica gel G plate; Developing solvent: cyclohexane extraction-ethyl acetate-diethylamine (16: 3: 1); Developer: bismuth potassium iodide test solution
Among Fig. 2: a-protopine, b-fibrauretin, c-Bicuculline, d-tetrahydropalmatine, e-bulbocapnine, f-dihydro Bicuculline
Liquid phase chromatogram condition: octadecylsilane chemically bonded silica is a filler; Mobile phase: second eyeball-methanol-0.1% phosphoric acid solution (15: 10: 75), transfer pH3.0 with triethylamine.
Detect wavelength: 282nm
The chromatographic peak result
| Name | Retention time (minute) | Area (microvolt * second) | Highly (microvolt) | Content | Unit | |
| 1 | 4.000 | 4625 | 941 | |||
| 2 | 5.733 | 23723 | 3467 | |||
| 3 | 7.181 | 10618 | 1269 | |||
| 4 | 8.114 | 12563 | 1428 | |||
| 5 | 9.493 | 192396 | 16378 | |||
| 6 | 11.047 | 84412 | 6707 | |||
| 7 | 16.191 | 217422 | 12088 |
The specific embodiment
Embodiment 1 preparation Hotseason grow corydaline
(1) get 1 kilogram of Hotseason grow tuber crude drug coarse powder, add 3 kilograms of soaked overnight of phosphoric acid solution of 2%, the diafiltration tube of packing into, the phosphoric acid solution diafiltration with 2% is collected percolate to alkaloid and is reacted till the reaction that is negative.Percolate is transferred about pH value to 6 with 10% sodium hydroxide, is concentrated into 1 gram crude drug/milliliter and gets final product.Use in order to separating;
(2) get concentrated solution, last bed volume is 2000 milliliters macroporous adsorbent resin DM
301, wash to color shallowly with the solution (2000-4000ml) of 45 ℃ of 2N NaOH, reuse distilled water (5000-10000ml) is washed till neutrality.Using 50% ethanol (1000-3000ml) washing again instead does not get off to there being Bicuculline;
(3) reuse contains 95% ethanol elution (2000-3000ml) of 0.3% phosphoric acid, collects eluent and is washed till negative get final product of alkaloid reaction, and it is pure to not having that eluent reclaims ethanol;
(4) transfer about PH to 12 with 10% sodium hydroxide solution, and add NaCl and make into the saturated common salt aqueous solution, place and make precipitation fully, collecting precipitation, saturated common salt aqueous solution be to neutral, drying and crushing.
(5) refining with crude product amount 4-8 dehydrated alcohol doubly, promptly get the Hotseason grow corydaline elaboration.
Precision takes by weighing protopine reference substance 10.0mg, puts in the 10ml measuring bottle, and it is an amount of to add methanol, and supersound process makes dissolving, put coldly, add methanol and be diluted to scale, shake up, promptly get reference substance solution (1.0mg/ml), precision is measured in 2ml to the 100ml measuring bottle, adds methanol and is diluted to scale, shakes up.Precision is measured above-mentioned reference substance solution 1ml, 2ml, and 3ml, 4ml, 5ml puts respectively in the 100ml tool plug conical flask, and water-bath volatilizes methanol.Accurate successively bromine potassium phenol green solution 2.0ml and the acetic acid-sodium acetate buffer solution 5.0ml of adding, 75 ℃ of heating in water bath 30 minutes, put cold, accurate chloroform 10ml, the tool plug of adding.Powerful jolting 2 minutes was placed 5 minutes, moved in the separatory funnel, and placement makes layering slightly, gets chloroform liquid, by a little funnel that the 2g anhydrous sodium sulfate is housed.Get chloroform liquid,, carry out spectral scan (slit 2.0, medium sweep) according to the test of an appendix VB of Chinese Pharmacopoeia version in 2000 spectrophotography.With corresponding solution is blank.Reference substance solution has absorption maximum at 415nm wavelength place as a result.With concentration is abscissa, and trap is a vertical coordinate, the drawing standard curve.
Get the about 17mg of this product, micrometric measurement is put in the 100ml measuring bottle, add 50% methanol supersound process and make dissolving and be diluted to scale, shake up, promptly, precision is measured above-mentioned need testing solution 0.50ml, put in the 100ml tool plug conical flask, the method under the preparation of sighting target directrix curve is from " adding bromine potassium phenol green solution 2.0ml successively ", measure trap at 415nm wavelength place in accordance with the law, read alkaloidal amount from standard curve, calculate, promptly getting the Hotseason grow total alkaloid content is 85.6%.
Selecting octadecylsilane chemically bonded silica with high performance liquid chromatography is filler AichromBond-AQ C
185 m (4.6 * 150mm) chromatographic columns (U.S.)
Detect wavelength: 290nm, flow velocity: 1.0ml/min, column temperature: 30 ℃
Mobile phase: acetonitrile-0.1% trifluoracetic acid (24: 76) is measured protopine content
Get the about 40mg of this product, the accurate title, decide, and puts in the 10ml measuring bottle, adds the mobile phase supersound process and make dissolving and be diluted to scale, shake up, precision is measured 1ml and is put in the 50ml measuring bottle, adds mobile phase and is diluted to scale, shakes up, filter with microporous filter membrane (0.45 μ m), get subsequent filtrate, get the raw material need testing solution.Precision takes by weighing protopine reference substance 5.00mg, puts in the 25ml measuring bottle, adds the mobile phase supersound process and makes dissolving and be diluted to scale, shakes up, and gets 5ml solution again to the 50ml measuring bottle, adds mobile phase and is diluted to scale, promptly gets reference substance solution and is (20 μ g/ml).Get each 20ul sample introduction of above-mentioned solution respectively, the record chromatogram, calculating and promptly getting protopine content is 38.1%.
Selecting octadecylsilane chemically bonded silica with high performance liquid chromatography is filler AichromBond-AQ C
185 m (4.6 * 150mm) chromatographic columns (U.S.)
Detect wavelength: 225nm, flow velocity: 1.0ml/min, column temperature: 30 ℃
Mobile phase: acetonitrile-0.1% trifluoracetic acid (24: 76) is measured Bicuculline content
Get the about 40mg of this product, the accurate title, decide, and puts in the 10ml measuring bottle, add the mobile phase supersound process and make dissolving and be diluted to scale, shake up, precision is measured 1ml and is put in the 50ml measuring bottle, add mobile phase and be diluted to scale, shake up, filter with microporous filter membrane (0.45 μ m), get subsequent filtrate, promptly getting the raw material need testing solution. precision takes by weighing Bicuculline reference substance 6.00mg, puts in the 10ml measuring bottle, adds the mobile phase supersound process and makes dissolving and be diluted to scale, shake up, promptly get Bicuculline stock solution (0.6mg/ml); Precision is measured in stock solution 1ml to the 100ml measuring bottle, adds mobile phase and is diluted to scale, shakes up, and promptly gets reference substance solution (6 μ g/ml).Get each 20ul sample introduction of above-mentioned solution respectively, the record chromatogram, calculating and promptly getting Bicuculline content is 3.6%.
Embodiment 2 preparation Hotseason grow corydalines
Use the used polar solvent of step (1) among the embodiment 1 instead 2% hydrochloric acid solution, other steps (2), (3), (4), (5) are fully with embodiment 1.
Calculating the Hotseason grow total alkaloids by content assaying method mensuration among the embodiment 1 is 84.2%, and protopine content is 35.6%, and Bicuculline content is 4.5%
Embodiment 3 preparation Hotseason grow corydalines
Use the used polar solvent of step (1) among the embodiment 1 instead 2% sulfuric acid solution, other steps (2), (3), (4), (5) are fully with embodiment 1.
Calculating the Hotseason grow total alkaloids by content assaying method mensuration among the embodiment 1 is 82.5%, and protopine content is 35.7%, and Bicuculline content is 4.8%
Embodiment 4 preparation Hotseason grow corydalines
Use the used polar solvent of step (1) among the embodiment 1 instead 2% acetum, other steps (2), (3), (4), (5) are fully with embodiment 1.
Calculating the Hotseason grow total alkaloids by content assaying method mensuration among the embodiment 1 is 83.4%, and protopine content is 35.1%, and Bicuculline content is 4.6%
Embodiment 5 preparation Hotseason grow corydalines
Step (1), (2), (3), (4), (5) are with embodiment 2, and wherein the macroporous adsorbent resin in the step (2) is used DM instead
201
Calculating the Hotseason grow total alkaloids by content assaying method mensuration among the embodiment 1 is 79.5%, and protopine content is 33.8%, and Bicuculline content is 5.1%.
Embodiment 6 preparation Hotseason grow corydalines
Step (1), (2), (3), (4), (5) are with embodiment 4, and wherein the macroporous adsorbent resin in the step (2) is used D instead
101A
Calculating the Hotseason grow total alkaloids by content assaying method mensuration among the embodiment 1 is 79.1%, and protopine content is 31.5%, and Bicuculline content is 4.2%.
Embodiment 7 preparation Hotseason grow corydalines
Step (1), (2), (3), (4), (5) are with embodiment 1, and wherein the macroporous adsorbent resin in the step (2) is used D instead
301
Calculating the Hotseason grow total alkaloids by content assaying method mensuration among the embodiment 1 is 80.9%, and protopine content is 36.3%, and Bicuculline content is 4.9%
Embodiment 8 preparation Hotseason grow corydalines
Step (1), (2), (3), (4), (5) are with embodiment 1, and wherein the polar solvent in the step (1) is used 95% alcoholic solution instead, and the macroporous adsorbent resin in the step (2) is used DM instead
201
Calculating the Hotseason grow total alkaloids by content assaying method mensuration among the embodiment 1 is 75.3%, and protopine content is 31.4%, and Bicuculline content is 4.2%.
Embodiment 9 preparation Hotseason grow corydalines
Step (1), (2), (3), (4), (5) are with embodiment 8, and wherein the polar solvent in the step (1) is used 95% methanol solution instead.
Calculating the Hotseason grow total alkaloids by content assaying method mensuration among the embodiment 1 is 74.8%, and protopine content is 32.6%, and Bicuculline content is 4.3%.
Preparation of embodiment 10 injection Hotseason grow corydalines and assay
Hotseason grow corydaline (embodiment 1) 2 grams
Mannitol 100g
Make 1000 bottles
Method for making: get Hotseason grow corydaline extract 2g, dissolve with 1%HCI solution, it is an amount of to add the injection water, stirring makes abundant dissolving, other gets mannitol 100g and adds in an amount of water for injection after the stirring and dissolving, merges two solution, is mixed and transfers PH to 3～5 with 1%NaOH solution, add an amount of water for injection again and be settled to 1000ml, add an amount of needle-use activated carbon stirring and boil 30min.The microporous filter membrane classification filters, and the filtrate quantitative aseptic is sub-packed in the good ampoule of aseptic process.Lyophilizing, jump a queue, roll lid promptly.Every bottle of specification is 2mg.Same preparation method, making every bottle of specification under the situation of rationally adjusting prescription is 1mg.
Assay:
Total alkaloids
Precision takes by weighing protopine reference substance 10.0mg, puts in the 10ml measuring bottle, and it is an amount of to add methanol, and supersound process makes dissolving, put coldly, add methanol and be diluted to scale, shake up, promptly get reference substance solution (1.0mg/ml), precision is measured in 2ml to the 100ml measuring bottle, adds methanol and is diluted to scale, shakes up.Precision is measured above-mentioned reference substance solution 1ml, 2ml, and 3ml, 4ml, 5ml puts respectively in the 100ml tool plug conical flask, and water-bath volatilizes methanol.Accurate successively bromine potassium phenol green solution 2.0ml and the acetic acid-sodium acetate buffer solution 5.0ml of adding, 75 ℃ of heating in water bath 30 minutes, put cold, accurate chloroform 10ml, the tool plug of adding.Powerful jolting 2 minutes was placed 5 minutes, moved in the separatory funnel, and placement makes layering slightly, gets chloroform liquid, by a little funnel that the 2g anhydrous sodium sulfate is housed.Get chloroform liquid,, carry out spectral scan (slit 2.0, medium sweep) according to the test of an appendix VB of Chinese Pharmacopoeia version in 2000 spectrophotography.With corresponding solution is blank.Reference substance solution has absorption maximum at 415nm wavelength place as a result.With concentration is abscissa, and trap is a vertical coordinate, the drawing standard curve.
Get this product number bottle, porphyrize is got about 0.2g, micrometric measurement is put in the 100ml measuring bottle, adds 50% methanol supersound process and makes dissolving and be diluted to scale, shake up, that is, precision is measured above-mentioned need testing solution 0.50ml, put in the 100ml tool plug conical flask, the method under the preparation of sighting target directrix curve is from " adding bromine potassium phenol green solution 2.0ml successively ", measure trap at 415nm wavelength place in accordance with the law, read alkaloidal amount from standard curve, calculate, promptly getting the Hotseason grow total alkaloid content is 85.1%.
Protopine
Selecting octadecylsilane chemically bonded silica is filler AichromBond-AQ C
185 m (4.6 * 150mm) chromatographic columns (U.S.)
Detect wavelength: 290nm, flow velocity: 1.0ml/min, column temperature: 30 ℃
Mobile phase: acetonitrile-0.1% trifluoracetic acid (24: 76)
Measure protopine content in the preparation according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D)
Get this product number bottle, grind well, get about 0.1g, the accurate title, decide, put in the 10ml measuring bottle, add the mobile phase supersound process and make dissolving and be diluted to scale, shake up, precision is measured 1ml and is put in the 50ml measuring bottle, add mobile phase and be diluted to scale, shake up, filter with microporous filter membrane (0.45 μ m), get subsequent filtrate, get the raw material need testing solution.Precision takes by weighing protopine reference substance 5.00mg, puts in the 25ml measuring bottle, adds the mobile phase supersound process and makes dissolving and be diluted to scale, shakes up, and gets 5ml solution again to the 50ml measuring bottle, adds mobile phase and is diluted to scale, promptly gets reference substance solution and is (20 μ g/ml).Get each 20ul sample introduction of above-mentioned solution respectively, the record chromatogram, calculating and promptly getting protopine content is 36.8%.
Bicuculline
Selecting octadecylsilane chemically bonded silica is filler AichromBond-AQ C
185 m (4.6 * 150mm) chromatographic columns (U.S.)
Detect wavelength: 225nm, flow velocity: 1.0ml/min, column temperature: 30 ℃
Mobile phase: acetonitrile-0.1% trifluoracetic acid (24: 76)
Measure Bicuculline content in the preparation according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D)
Get this product number bottle, grind well, get about 0.1g, the accurate title, decide, put in the 10ml measuring bottle, add the mobile phase supersound process and make dissolving and be diluted to scale, shake up, precision is measured 1ml and is put in the 50ml measuring bottle, add mobile phase and be diluted to scale, shake up, filter with microporous filter membrane (0.45 μ m), get subsequent filtrate, promptly getting the raw material need testing solution. precision takes by weighing Bicuculline reference substance 6.00mg, puts in the 10ml measuring bottle, adds the mobile phase supersound process and makes dissolving and be diluted to scale, shake up, promptly get Bicuculline stock solution (0.6mg/ml); Precision is measured in stock solution 1ml to the 100ml measuring bottle, adds mobile phase and is diluted to scale, shakes up, and promptly gets reference substance solution (6 μ g/ml).Get each 20ul sample introduction of above-mentioned solution respectively, the record chromatogram, calculating and promptly getting Bicuculline content is 3.8%.
Embodiment 11 (preparation of injection Hotseason grow corydaline and assay):
Hotseason grow corydaline (embodiment 1) 1 gram (2g)
Mannitol 50 grams
Make 1000 bottles
Method for making: get Hotseason grow corydaline extract 2g, use 1%H
3PO
4The solution dissolving, it is an amount of to add the injection water, stirring makes abundant dissolving, other gets mannitol 50g and adds in an amount of water for injection after the stirring and dissolving, merge two solution, be mixed and transfer PH to 3～5 to add an amount of water for injection again and be settled to 1000ml, add an amount of needle-use activated carbon stirring and boil 30min with 1%NaOH solution.The microporous filter membrane classification filters, and the filtrate quantitative aseptic is sub-packed in the good ampoule of aseptic process.Lid is jumped a queue, rolled to lyophilizing promptly.Every bottle of specification is 1mg or 2mg.
Assay:
Total alkaloids
Precision takes by weighing protopine reference substance 10.0mg, puts in the 10ml measuring bottle, and it is an amount of to add methanol, and supersound process makes dissolving, put coldly, add methanol and be diluted to scale, shake up, promptly get reference substance solution (1.0mg/ml), precision is measured in 2ml to the 100ml measuring bottle, adds methanol and is diluted to scale, shakes up.Precision is measured above-mentioned reference substance solution 1ml, 2ml, and 3ml, 4ml, 5ml puts respectively in the 100ml tool plug conical flask, and water-bath volatilizes methanol.Accurate successively bromine potassium phenol green solution 2.0ml and the acetic acid-sodium acetate buffer solution 5.0ml of adding, 75 ℃ of heating in water bath 30 minutes, put cold, accurate chloroform 10ml, the tool plug of adding.Powerful jolting 2 minutes was placed 5 minutes, moved in the separatory funnel, and placement makes layering slightly, gets chloroform liquid, by a little funnel that the 2g anhydrous sodium sulfate is housed.Get chloroform liquid,, carry out spectral scan (slit 2.0, medium sweep) according to the test of an appendix V of Chinese Pharmacopoeia version in 2000 B spectrophotography.With corresponding solution is blank.Reference substance solution has absorption maximum at 415nm wavelength place as a result.With concentration is abscissa, and trap is a vertical coordinate, the drawing standard curve.
Get this product number bottle, porphyrize is got about 0.2g, micrometric measurement is put in the 100ml measuring bottle, adds 50% methanol supersound process and makes dissolving and be diluted to scale, shake up, that is, precision is measured above-mentioned need testing solution 0.50ml, put in the 100ml tool plug conical flask, the method under the preparation of sighting target directrix curve is from " adding bromine potassium phenol green solution 2.0ml successively ", measure trap at 415nm wavelength place in accordance with the law, read alkaloidal amount from standard curve, calculate, promptly getting the Hotseason grow total alkaloid content is 86.3%.
Protopine
Selecting octadecylsilane chemically bonded silica is filler AichromBond-AQ C
185 m (4.6 * 150mm) chromatographic columns (U.S.)
Detect wavelength: 290nm, flow velocity: 1.0ml/min, column temperature: 30 ℃
Mobile phase: acetonitrile-0.1% trifluoracetic acid (24: 76)
Measure protopine content in the preparation according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D)
Get this product number bottle, grind well, get about 0.1g, the accurate title, decide, put in the 10ml measuring bottle, add the mobile phase supersound process and make dissolving and be diluted to scale, shake up, precision is measured 1ml and is put in the 50ml measuring bottle, add mobile phase and be diluted to scale, shake up, filter with microporous filter membrane (0.45 μ m), get subsequent filtrate, get the raw material need testing solution.Precision takes by weighing protopine reference substance 5.00mg, puts in the 25ml measuring bottle, adds the mobile phase supersound process and makes dissolving and be diluted to scale, shakes up, and gets 5ml solution again to the 50ml measuring bottle, adds mobile phase and is diluted to scale, promptly gets reference substance solution and is (20 μ g/ml).Get each 20ul sample introduction of above-mentioned solution respectively, the record chromatogram, calculating and promptly getting protopine content is 37.6%.
Bicuculline
Selecting octadecylsilane chemically bonded silica is filler AichromBond-AQ C
185 m (4.6 * 150mm) chromatographic columns (U.S.)
Detect wavelength: 225nm, flow velocity: 1.0ml/min, column temperature: 30 ℃
Mobile phase: acetonitrile-0.1% trifluoracetic acid (24: 76)
Measure Bicuculline content in the preparation according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 D)
Get this product number bottle, grind well, get about 0.1g, the accurate title, decide, put in the 10ml measuring bottle, add the mobile phase supersound process and make dissolving and be diluted to scale, shake up, precision is measured 1ml and is put in the 50ml measuring bottle, add mobile phase and be diluted to scale, shake up, filter with microporous filter membrane (0.45 μ m), get subsequent filtrate, promptly getting the raw material need testing solution. precision takes by weighing Bicuculline reference substance 6.00mg, puts in the 10ml measuring bottle, adds the mobile phase supersound process and makes dissolving and be diluted to scale, shake up, promptly get Bicuculline stock solution (0.6mg/ml); Precision is measured in stock solution 1ml to the 100ml measuring bottle, adds mobile phase and is diluted to scale, shakes up, and promptly gets reference substance solution (6 μ g/ml).Get each 20ul sample introduction of above-mentioned solution respectively, the record chromatogram, calculating and promptly getting Bicuculline content is 3.7%.
Embodiment 12 mouse tail vein injections and the acute toxicity test of lumbar injection injection Hotseason grow
Summary: this experimentation the acute toxicity of mouse tail vein injection and lumbar injection injection Hotseason grow.The result shows that the LD50 of mouse tail vein injection injection Hotseason grow is 6.889mg/kg, and 95% fiducial limit is 6.475～7.315mg/kg; The mouse peritoneal injection injection is 48.015mg/kg with the LD50 of Hotseason grow, and 95% fiducial limit is 44.886～51.332mg/kg.
Purpose: after observing mouse tail vein injection and lumbar injection injection Hotseason grow, acute toxic reaction that is produced and death condition are measured the LD50 value, for clinical practice provides the safety foundation.
1 experiment material
1.1 120 of animal NIH mices, male and female half and half, Department of Health of Guangdong Province's Experimental Animal Center provides, the quality certification number: SCXK (Guangdong) 2003-0002, word 2005A011 checks and affirm in Guangdong.
1.2 medicine injection Hotseason grow derives from embodiment 10, is diluted to desired concn with normal saline before the test.The injection normal saline, Guangxi Yu Yuan pharmaceutcal corporation, Ltd, lot number C20050520.
2. method
2.1 tail intravenously administrable acute toxicity is got 60 of body weight 18～22g NIH mices, male and female half and half are divided into 6 groups at random, be I, II, III, IV, (dosage is respectively 10.00mg/kg to the V group, 8.50mg/kg, 7.22mg/kg, 6.14mg/kg, 5.22mg/kg) with the blank group, 10 every group.The tail vein injection administration, the administration capacity is the 0.2ml/10g body weight, and the blank group is given the normal saline of the capacity of grade.Observe mice body weight change, diet, outward appearance, behavior, secretions, Excreta, death condition and toxic reaction (symptom of toxic reaction, the order of severity, zero-time, persistent period, whether reversible) etc., 14 days observing times after the administration.Dying and dead animal are in time performed an autopsy on sb, and other animals perform an autopsy on sb after the observation period finishes, and observe internal organs volume, color and quality and have or not change, if there is the visible pathological changes of naked eyes need carry out histopathologic examination.
2.2 the intraperitoneal injection acute toxicity is got 60 of body weight 18～22g NIH mices, male and female half and half, be divided into 5 groups at random, be I, II, III, IV, (dosage is respectively 60.00mg/kg to the V group, 54.00mg/kg, 48.60mg/kg, 43.74mg/kg, 39.37mg/kg) and the blank group, 10 every group.Intraperitoneal injection, the administration capacity is the 0.2ml/10g body weight, and the blank group is given the normal saline of the capacity of grade.Observe mice body weight change, diet, outward appearance, behavior, secretions, Excreta, death condition and toxic reaction (symptom of toxic reaction, the order of severity, zero-time, persistent period, whether reversible) etc., 14 days observing times after the administration.Dying and dead animal are in time performed an autopsy on sb, and other animals perform an autopsy on sb after the observation period finishes, and observe internal organs volume, color and quality and have or not change, if there is the visible pathological changes of naked eyes need carry out histopathological examination.
3 date processing
Data statistics adopts new drug statistical software (NDST, 5.1 versions) to handle, and the Bliss method is calculated the fiducial limit of LD50 and 95%.
4 results
4.1 behind the tail intravenously administrable acute toxicity mouse tail vein injection injection Hotseason grow, as seen dosage correlation movable reduces, perpendicular hair, licks the friction mutually of foot, forelimb, rapid breathing, sialorrhea, urinary incontinence, convulsions, tic, muscle rigidity contraction, jumping characteristic death occurs at last, animal dead starts from after the administration 5 seconds, and animal dead betides after the administration in the 1min mostly.The animal toxic reaction does not have obviously relevant with mortality rate and sex.Mice shows no obvious abnormalities the normal saline group.Dead animal and observation period finish to put to death the animal via postmortem, and organs such as the perusal heart, liver, spleen, lung, kidney, brain are not seen obvious pathological change.The results are shown in Table 1.
Table 1 injection Hotseason grow tail vein injection administration acute toxicity testing result
| Group | Dosage (mg/kg) | Number of animals (only) | Dead animal number (only) | Mortality rate | Probit (y) |
| I | 10.00 | 10 | 10 | 1.0 | 6.96 |
| II | 8.50 | 10 | 9 | 0.9 | 6.28 |
| III | 7.22 | 10 | 6 | 0.6 | 5.25 |
| IV | 6.14 | 10 | 3 | 0.3 | 4.48 |
| V | 5.22 | 10 | 0 | 0 | 3.04 |
| The blank group | Normal saline | 10 | 0 | 0 |
LD50 is 6.889mg/kg, and 95% fiducial limit is 6.475～7.315mg/kg;
4.2 intraperitoneal injection acute toxicity mouse peritoneal injection injection with Hotseason grow after, as seen the dosage correlation activity reduces, erects hair, licks the friction mutually of foot, forelimb, rapid breathing, sialorrhea, urinary incontinence, convulsions, tic, jumping characteristic death, animal dead starts from after the administration 2 minutes, and animal dead betides after the administration in 1 hour mostly.The animal toxic reaction does not have obviously relevant with mortality rate and sex.Mice shows no obvious abnormalities the normal saline group.Dead animal and observation period finish to put to death the animal via postmortem, and organs such as the perusal heart, liver, spleen, lung, kidney, brain are not seen obvious pathological change.The results are shown in Table 2.
Table 2 injection Hotseason grow intraperitoneal injection acute toxicity testing result
| Group | Dosage (mg/kg) | Number of animals (only) | Dead animal number (only) | Mortality rate | Probit (y) |
| I | 60.00 | 10 | 10 | 1.0 | 6.96 |
| II | 54.00 | 10 | 9 | 0.9 | 6.28 |
| III | 48.60 | 10 | 4 | 0.4 | 4.75 |
| IV | 43.74 | 10 | 3 | 0.3 | 4.48 |
| V | 39.37 | 10 | 0 | 0 | 3.04 |
| The blank group | Normal saline | 10 | 0 | 0 |
LD50 is 48.015mg/kg, and 95% fiducial limit is 44.886～51.332mg/kg.
5 interpretations of result
5.1 the LD50 of mouse tail vein injection injection Hotseason grow is 6.889mg/kg, calculates by kg body weight, is equivalent to more than 120 times of 70kg adult's consumption every day (0.057mg/kg).
5.2 the mouse peritoneal injection injection is 48.015mg/kg with the LD50 of Hotseason grow, calculates by kg body weight, is equivalent to more than 842 times of 70kg adult's intravenously administrable amount every day (0.057mg/kg).
6 conclusions
The LD50 of mouse tail vein injection injection Hotseason grow is 6.889mg/kg, and 95% fiducial limit is 6.475～7.315mg/kg; The mouse peritoneal injection injection is 48.015mg/kg with the LD50 of Hotseason grow, and 95% fiducial limit is 44.886～51.332mg/kg.
List of references:
1. State Food and Drug Administration's medicine is evaluated the center. Chinese medicine, natural drug studies on acute toxicity technological guidance principle .2005.3
2. king north baby, Li Yikui. new Chinese medicine is developed technology and method. Shanghai: Shanghai science tech publishing house, 2002:788～790.
3. bureau of drug policy ﹠ administration of Ministry of Health of the People's Republic of China. study of tcm new drug guide .1993:203.
Embodiment 13, rat tail vein injection and the acute toxicity test of lumbar injection injection Hotseason grow
Summary: this experimentation the acute toxicity of rat tail vein injection and lumbar injection injection Hotseason grow.The result shows that the rat tail vein injection injection is 8.617mg/kg with the LD50 of Hotseason grow, and 95% fiducial limit is 8.258～9.104mg/kg; The LD50 of rats by intraperitoneal injection injection Hotseason grow is 55.079mg/kg, and 95% fiducial limit is 52.302～58.733mg/kg.
Purpose: after observing rat tail vein injection and lumbar injection injection Hotseason grow, acute toxic reaction that is produced and death condition are measured the LD50 value, for clinical practice provides the safety foundation.
1 experiment material
1.1 120 of animal SD rats, male and female half and half, Department of Health of Guangdong Province's Experimental Animal Center provides, the quality certification number: SCXK (Guangdong) 2003-0002, word 2005A010 checks and affirm in Guangdong.
1.2 medicine injection Hotseason grow derives from embodiment 10, is diluted to desired concn with normal saline before the test.The injection normal saline, Guangxi Yu Yuan pharmaceutcal corporation, Ltd, lot number C20050520.
2. method
2.1 tail intravenously administrable acute toxicity is got 60 of body weight 130～150g SD rats, male and female half and half are divided into 6 groups at random, be I, II, III, IV, (dosage is respectively 12.00mg/kg to the V group, 9.60mg/kg, 7.68mg/kg, 6.14mg/kg, 4.92mg/kg) with the blank group, 10 every group.The tail vein injection administration, the administration capacity is the 0.5ml/100g body weight, and the blank group is given the normal saline of the capacity of grade.Observe rat body weight variation, diet, outward appearance, behavior, secretions, Excreta, death condition and toxic reaction (symptom of toxic reaction, the order of severity, zero-time, persistent period, whether reversible) etc., 14 days observing times after the administration.Dying and dead animal are in time performed an autopsy on sb, and other animals perform an autopsy on sb after the observation period finishes, and observe internal organs volume, color and quality and have or not change, if there is the visible pathological changes of naked eyes need carry out histopathologic examination.
2.2 the intraperitoneal injection acute toxicity is got 60 of body weight 130～150g SD rats, male and female half and half, be divided into 6 groups at random, be I, II, III, IV, (dosage is respectively 70.00mg/kg to the V group, 59.50mg/kg, 50.58mg/kg, 42.99mg/kg, 36.54mg/kg) and the blank group, 10 every group.Intraperitoneal injection, the administration capacity is the 0.5ml/100g body weight, and the blank group is given the normal saline of the capacity of grade.Observe rat body weight variation, diet, outward appearance, behavior, secretions, Excreta, death condition and toxic reaction (symptom of toxic reaction, the order of severity, zero-time, persistent period, whether reversible) etc., 14 days observing times after the administration.Dying and dead animal are in time performed an autopsy on sb, and other animals perform an autopsy on sb after the observation period finishes, and observe internal organs volume, color and quality and have or not change, if there is the visible pathological changes of naked eyes need carry out histopathological examination.
3 date processing
Data statistics adopts new drug statistical software (NDST, 5.1 versions) to handle, and the Bliss method is calculated the fiducial limit of LD50 and 95%.
4 results
4.1 tail intravenously administrable acute toxicity rat tail vein injection injection with Hotseason grow after, as seen dosage correlation movable reduces, perpendicular hair, licks that the friction mutually of foot, forelimb, rapid breathing, sialorrhea, eye socket are hemorrhage, urinary incontinence, convulsions, tic, muscle rigidity contraction, jumping characteristic death occurs at last, animal dead starts from after the administration 25 seconds, and animal dead betides after the administration in 30 minutes mostly.The animal toxic reaction does not have obviously relevant with mortality rate and sex.Rat shows no obvious abnormalities the normal saline group.Dead animal and observation period finish to put to death the animal via postmortem, and organs such as the perusal heart, liver, spleen, lung, kidney, brain are not seen obvious pathological change.The results are shown in Table 1.
Table 1 injection Hotseason grow rat tail vein drug administration by injection acute toxicity testing result
| Group | Dosage (mg/kg) | Number of animals (only) | Dead animal number (only) | Mortality rate | Probit (y) |
| I | 12.00 | 10 | 9 | 0.9 | 6.28 |
| II | 9.60 | 10 | 7 | 0.7 | 5.52 |
| III | 7.68 | 10 | 3 | 0.3 | 4.48 |
| IV | 6.14 | 10 | 1 | 0.1 | 3.72 |
| V | 4.92 | 10 | 0 | 0 | 3.04 |
| The blank group | Normal saline | 10 | 0 | 0 |
LD50 is 8.617mg/kg, and 95% fiducial limit is 8.258～9.104mg/kg;
4.2 behind the intraperitoneal injection acute toxicity rats by intraperitoneal injection injection Hotseason grow, as seen dosage correlation movable reduce, perpendicular hair, lick that the friction mutually of foot, forelimb, rapid breathing, sialorrhea, eye socket are hemorrhage, urinary incontinence, convulsions, tic, jumping characteristic death, animal dead starts from after the administration 5 minutes, and animal dead betides after the administration in 1 hour mostly.The animal toxic reaction does not have obviously relevant with mortality rate and sex.Rat shows no obvious abnormalities the normal saline group.Dead animal and observation period finish to put to death the animal via postmortem, and organs such as the perusal heart, liver, spleen, lung, kidney, brain are not seen obvious pathological change.The results are shown in Table 2.
Table 2 injection Hotseason grow rats by intraperitoneal injection administration acute toxicity testing result
| Group | Dosage (mg/kg) | Number of animals (only) | Dead animal number (only) | Mortality rate | Probit (y) |
| I | 70.00 | 10 | 9 | 0.9 | 6.28 |
| II | 59.50 | 10 | 6 | 0.6 | 5.25 |
| III | 50.58 | 10 | 4 | 0.4 | 4.75 |
| IV | 42.99 | 10 | 1 | 0.1 | 3.72 |
| V | 36.54 | 10 | 0 | 0 | 3.04 |
| The blank group | Normal saline | 10 | 0 | 0 |
LD50 is 55.079mg/kg, and 95% fiducial limit is 52.302～58.733mg/kg.
5 interpretations of result
5.1 the rat tail vein injection injection is 8.617mg/kg with the LD50 of Hotseason grow, calculates by kg body weight, is equivalent to more than 150 times of 70kg adult's consumption every day (0.057mg/kg).
5.2 the LD50 of rats by intraperitoneal injection injection Hotseason grow is 55.079mg/kg, calculates by kg body weight, is equivalent to more than 966 times of 70kg adult's intravenously administrable amount every day (0.057mg/kg).
6 conclusions
The rat tail vein injection injection is 8.617mg/kg with the LD50 of Hotseason grow, and 95% fiducial limit is 8.258～9.104mg/kg; The LD50 of rats by intraperitoneal injection injection Hotseason grow is 55.079mg/kg, and 95% fiducial limit is 52.302～58.733mg/kg.
List of references:
1. State Food and Drug Administration's medicine is evaluated the center. Chinese medicine, natural drug studies on acute toxicity technological guidance principle .2005.3
2. king north baby, Li Yikui. new Chinese medicine is developed technology and method. Shanghai: Shanghai science tech publishing house, 2002:788～790.
3. bureau of drug policy ﹠ administration of Ministry of Health of the People's Republic of China. study of tcm new drug guide .1993:203.
Claims (11)
1, a kind of Hotseason grow corydaline, extract by the Hotseason grow dry tuber, it is characterized in that by weight percentage, total alkali wherein contains protopine (protopine) 35-70%, Bicuculline (bicuculline)≤5%, tetrahydropalmatine (tetrahydropalmatine), fibrauretin (palmatine), papaverine, dihydro Bicuculline, bulbocapnine, bulbocapnine etc. more than 80%.
2, a kind of preparation method of Hotseason grow corydaline is characterized in that:
(1) be raw material with the Hotseason grow tuber, the polar solvent percolation that adds 2-20 times extracts, or the forced refluence extraction, collects extracting solution to the inanimate object alkali reaction, and merge extractive liquid, is transferred PH to 6-7 with alkali liquor, is concentrated in right amount, obtains extracting concentrated solution;
(2) the extraction concentrated solution that above-mentioned steps is obtained carries out the macroporous resin adsorption chromatography, and with the 1-8mol/l strong base solution washing of temperature more than 40 ℃, consumption is 1-10 a times of column volume, washing roguing of reuse 50-60% alcoholic solution and hypertoxicity composition Bicuculline;
(3) with polarity eluent eluting Hotseason grow alkaloid, collect eluent, reclaim solvent, be concentrated into small size;
(4) transfer pH value to alkalescence with alkali liquor, separate out precipitation, the leaching precipitation, saturated common salt is washed to neutrality, drying;
(5) precipitation is filtered with organic solvent ethanol or dissolve with methanol, and filtrate is reclaimed solvent to small size, places crystallize, and the leaching crystallizing and drying promptly gets Hotseason grow corydaline.
3, the preparation method of Hotseason grow corydaline according to claim 2 is characterized in that used polar solvent in the step (1) is selected from phosphoric acid, hydrochloric acid, citric acid, tartaric acid or the succinic acid solution of 1-10%, or the ethanol of 75-95% or methanol solution.
4, the preparation method of Hotseason grow corydaline according to claim 2 is characterized in that big pore adsorption resin is D in the step (2)
101A, DM
201, DM
301, D
301In a kind of.
5, the preparation method of Hotseason grow corydaline according to claim 2, it is characterized in that the polarity eluent sour water that is used for the eluting Hotseason grow corydaline used in the step (3) is hydrochloric acid, phosphoric acid, lactic acid or the succinic acid solution of 0.1-10%, or concentration is acidic ethanol or the acidic methanol solution of 60-95%.
6, the preparation method of Hotseason grow corydaline according to claim 2 is characterized in that used alkali liquor is that concentration is NaOH or the KOH solution of 5-20% in the step (4).
7, the preparation method of Hotseason grow corydaline according to claim 2, it is characterized in that in the step (5) used organic solvent be chloroform or≤C4 lower aliphatic alcohols, acetone or ethyl acetate.
8, a kind of pharmaceutical composition that contains Hotseason grow corydaline is characterized in that dosing conventional pharmaceutical excipient, promptly can be made into oral, injection type.
9, a kind of pharmaceutical composition that contains Hotseason grow corydaline, it is characterized in that the Hotseason grow corydaline freeze-dried powder, prescription is formed: Hotseason grow corydaline 1～5g, 0.5～10% hydrochloric acid, phosphoric acid, lactic acid or succinic acid solution 5～10ml, in excipient mannitol, dextran, glucose or the sodium chloride one or both or two or more 20～5000g add the injection water and make 1000ml in right amount.
10, the pharmaceutical composition that contains Hotseason grow corydaline according to claim 9 is characterized in that Hotseason grow corydaline 2g, 1% phosphoric acid solution 10ml, and mannitol 50g adds the injection water and makes 1000ml in right amount.
11, the Hotseason grow corydaline preparation of drug combination method that contains according to claim 9, it is characterized in that getting the Hotseason grow corydaline extract, dissolve with acid solution, it is an amount of to add the injection water, stirring makes abundant dissolving, other gets mannitol and adds in an amount of water for injection after the stirring and dissolving, merge two solution, be mixed and transfer PH to 3～5, add an amount of water for injection and standardize solution again, add an amount of needle-use activated carbon stirring and boiled 30 minutes with 1%NaOH solution, the microporous filter membrane classification filters, the filtrate quantitative aseptic is sub-packed in the good control antibiotic glass bottle of aseptic process, and vacuum lyophilization is jumped a queue, roll lid promptly.
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Cited By (7)
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|---|---|---|---|---|
| CN101829195A (en) * | 2010-04-26 | 2010-09-15 | 吉林大学 | Corydalis ochotensis total alkaloid and preparation method and medicinal application thereof |
| CN102475743A (en) * | 2010-11-26 | 2012-05-30 | 上海医药工业研究院 | Decumbent corydalis tuber total alkaloid refined substance, its preparation method and its application |
| CN103356752A (en) * | 2013-08-02 | 2013-10-23 | 江西天施康中药股份有限公司 | Corydalisdecumbens(Thunb.)Pers. injection and preparation method thereof |
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| CN103623066A (en) * | 2013-12-12 | 2014-03-12 | 山东阿如拉药物研究开发有限公司 | Corydalis impatiens total alkaloid extractive for preparing anti-cancer drugs and application thereof |
| CN107674081A (en) * | 2017-10-12 | 2018-02-09 | 成都瑞芬思生物科技有限公司 | A kind of preparation method of bicuculline and Biflorine |
| CN111948203A (en) * | 2019-08-20 | 2020-11-17 | 江西天施康中药股份有限公司 | Detection method of corydalis amabilis alkaloids |
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| CN101829195A (en) * | 2010-04-26 | 2010-09-15 | 吉林大学 | Corydalis ochotensis total alkaloid and preparation method and medicinal application thereof |
| CN101829195B (en) * | 2010-04-26 | 2012-07-04 | 吉林大学 | Preparation method of corydalis ochotensis total alkaloid |
| CN102475743A (en) * | 2010-11-26 | 2012-05-30 | 上海医药工业研究院 | Decumbent corydalis tuber total alkaloid refined substance, its preparation method and its application |
| CN103356752A (en) * | 2013-08-02 | 2013-10-23 | 江西天施康中药股份有限公司 | Corydalisdecumbens(Thunb.)Pers. injection and preparation method thereof |
| CN103610762A (en) * | 2013-12-12 | 2014-03-05 | 山东阿如拉药物研究开发有限公司 | Extract of corydalis impatiens total alkaloids and extraction method thereof |
| CN103623066A (en) * | 2013-12-12 | 2014-03-12 | 山东阿如拉药物研究开发有限公司 | Corydalis impatiens total alkaloid extractive for preparing anti-cancer drugs and application thereof |
| CN103610762B (en) * | 2013-12-12 | 2015-04-29 | 山东金诃药物研究开发有限公司 | Extract of corydalis impatiens total alkaloids and extraction method thereof |
| CN103623066B (en) * | 2013-12-12 | 2015-07-01 | 山东金诃药物研究开发有限公司 | Corydalis impatiens total alkaloid extractive for preparing anti-cancer drugs and application thereof |
| CN107674081A (en) * | 2017-10-12 | 2018-02-09 | 成都瑞芬思生物科技有限公司 | A kind of preparation method of bicuculline and Biflorine |
| CN107674081B (en) * | 2017-10-12 | 2019-12-31 | 成都瑞芬思生物科技有限公司 | Preparation method of bicuculline and protopine |
| CN111948203A (en) * | 2019-08-20 | 2020-11-17 | 江西天施康中药股份有限公司 | Detection method of corydalis amabilis alkaloids |
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