CN100376596C - Buxine, buxine hydrochloride, and its preparing method and formulation - Google Patents
Buxine, buxine hydrochloride, and its preparing method and formulation Download PDFInfo
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- CN100376596C CN100376596C CNB2006100575432A CN200610057543A CN100376596C CN 100376596 C CN100376596 C CN 100376596C CN B2006100575432 A CNB2006100575432 A CN B2006100575432A CN 200610057543 A CN200610057543 A CN 200610057543A CN 100376596 C CN100376596 C CN 100376596C
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Abstract
The present invention discloses buxine which is basically composed of cyclovirobuxine D, ring buxine D and ring evergreen buxine C. The weight content of the cyclovirobuxine D, the weight content of the ring buxine D and the weight content of the ring evergreen buxine C respectively are 60 to 95 %, 1.0 to 30% and 0.5 to 20%. The present invention also discloses hydrochloride of the buxine, a preparation method thereof and a preparation which is made from the buxine or the hydrochloride of the buxine. The composition and the structure of the buxine of the present invention and the hydrochloride of the buxine are clear, and effective quality control can be carried out. Therefore, the stable therapeutic effect is ensured, various modern preparations are easily made, the preparation technology is simple, and the cost is low.
Description
Technical field
The present invention relates to extraction separation and alkaloid valid target-" buxine " cyclovirobuxinum D, cyclobuxine D and three kinds of main alkaloid compositions of Cyclovirobuxine C, that have collaborative cardiovascular and cerebrovascular diseases effect that contain for preparing from Ramulus Buxi Sinicae-little leaf boxwood, and the raw material of hydrochloride and their pharmaceutical preparation, and their preparation method and mass analysis control method.
Background technology
Ramulus Buxi Sinicae is as medicinal, for book on Chinese herbal medicine records.Cyclovirobuxinum D is for separating the alkaloid monomer that obtains in Ramulus Buxi Sinicae, cyclovirobuxinum D raw material and preparation thereof are by " Chinese pharmacopoeia one one of version in 2005 is recorded " Buxine Tablet " that is respectively " cyclovirobuxinum D " raw material and is made by the cyclovirobuxinum D raw material.What the raw material of the cyclovirobuxinum D of putting down in writing in the pharmacopeia and Buxine Tablet adopted is the cyclovirobuxinum D monomer.Be used for the treatment of cardiovascular disordeies such as the coronary heart disease relevant, heart disorder clinically with diseases such as the Obstruction of qi in the chest and cardialgia of caused by energy stagnation and blood stasis, intermittent pulse.
Alkaloid in the Ramulus Buxi Sinicae has nearly 80 kinds (beam is grasped Wen Zhangchao position " buxine and cardiovascular and cerebrovascular diseases ", blue sky press 2000.5).Three kinds of compositions in the alkaloid that cyclovirobuxinum D (Cyclovirobuxine D), cyclobuxine D (Cyclobuxine D) and three kinds of alkaloids of Cyclovirobuxine C (Cyclovirobuxine C) all are Ramulus Buxi Sinicae.
The chemical molecular formula of cyclovirobuxinum D (Cyclovirobuxine D) is C 26H 46N 2O, its molecular weight are 402.36, are the off-white color crystalline powder, and easily molten in the chloroform, insoluble in the water, structural formula is as follows:
The chemical molecular formula of cyclobuxine D (Cyclobuxine D) is C 25H 42N 2O, molecular weight are 386.36, are off-white powder, and be easily molten in the chloroform, insoluble in the water, structural formula as
Down:
The molecular formula of Cyclovirobuxine C (Cyclovirobuxine C) is C 27H 48N 2O, molecular weight are 416.37, are the off-white color crystalline powder, and chemical structural formula is:
Cyclovirobuxinum D and other alkaloid are insoluble in water, the monomeric purification ratio of cyclovirobuxinum D is difficulty, complex process, cost height, though cyclovirobuxinum D raw material and preparation thereof are by " Chinese pharmacopoeia one one of version in 2005 is recorded, but, cause the medicine cost height that adopts the preparation of cyclovirobuxinum D monomer owing to adopted the cyclovirobuxinum D monomer to make raw material.And the effective drug duration of the medicine that employing cyclovirobuxinum D monomer is made is short.
Summary of the invention
The high problem of cost that the medicine preparation of making at present cyclovirobuxinum D raw material monomer and by this raw material monomer exists the object of the present invention is to provide a kind of buxine, and it is low to extract easy cost, and has collaborative cardiovascular disease therapies activity.
Another object of the present invention is to provide a kind of buxine hydrochloride, to improve the solvability of buxine composition.
The 3rd purpose of the present invention is to provide the preparation method of a kind of buxine, buxine hydrochloride.
The 4th purpose of the present invention then is to provide the preparation of a kind of buxine, buxine hydrochloride.
In order to realize first purpose of the present invention, buxine provided by the invention adopts following technical scheme, a kind of buxine, substantially be made up of cyclovirobuxinum D, cyclobuxine D and Cyclovirobuxine C, its weight content is respectively
cyclovirobuxinum D
60%～95%, cyclobuxine D 1.0%～30%, Cyclovirobuxine C 0.5%～20%.
Can also contain weight content in the buxine and be the other biological alkali below 10%.
Because the alkaloid in the buxine is insoluble in water, be difficult for being absorbed by human body, for improving its solubility property so that make the preparation that easily is absorbed by the body, described buxine is made hydrochloride, to realize second purpose of the present invention, its technical scheme is: a kind of buxine hydrochloride, described buxine hydrochloride is substantially by the hydrochloride of cyclovirobuxinum D, the hydrochloride of cyclobuxine D and the hydrochloride of Cyclovirobuxine C are formed, and its weight content is respectively the
hydrochloride
60%～95% of cyclovirobuxinum D, the hydrochloride 1.0%～30% of cyclobuxine D, the hydrochloride 0.5%～20% of Cyclovirobuxine C.
As the 3rd purpose of the present invention, the present invention also provides a kind of simple and easy to do method by Ramulus Buxi Sinicae extraction preparation buxine of the present invention: Ramulus Buxi Sinicae is ground into powder, adds 8-12 and doubly measure the ammonia soln immersion, add chloroform and extract, with chloroform extracted solution reclaim under reduced pressure chloroform, get residue; Add sherwood oil and disperse residue, adding dilute hydrochloric acid solution extracts, extracting solution with activated carbon decolorizing after, add in the ammoniacal liquor and dilute hydrochloric acid solution to alkalescence, use chloroform extraction again, with the most of solvent of chloroform extracted solution pressure reducing and steaming, when having treated that a large amount of solids are separated out, reflux also adds chloroform and just makes dissolving in right amount, and place at cold then place, separate out crystallization, filter; With the chloroform recrystallization at least once, drying under reduced pressure gets off-white color to faint yellow buxine crystallization.
The buxine hydrochloride is then made by alkaloid and hcl reaction, its preparation method is: the buxine that will be made up of cyclovirobuxinum D, cyclobuxine D and Cyclovirobuxine C substantially is dissolved in the chloroform, under stirring state, in chloroformic solution, feed excessive dry hydrogen chloride gas, generation is insoluble to the buxine hydrochloride precipitation of chloroform, after reaction is finished, leach precipitation, precipitate with washing with alcohol, through drying under reduced pressure, with ethanol-chloroform recrystallization, drying under reduced pressure obtains the buxine hydrochloride to constant weight again.
The present invention also provides a kind of buxine preparation, and said preparation is made by buxine or its hydrochloride;
Described buxine is made up of cyclovirobuxinum D, cyclobuxine D and Cyclovirobuxine C substantially, and its weight content is respectively
cyclovirobuxinum D
60%～95%, cyclobuxine D1.0%～30%, Cyclovirobuxine C 0.5%～20%;
Described buxine hydrochloride is made up of the hydrochloride of cyclovirobuxinum D, the hydrochloride of cyclobuxine D and the hydrochloride of Cyclovirobuxine C substantially, and its weight content is respectively
cyclovirobuxinum D hydrochloride
60%～95%, cyclobuxine D hydrochloride 1.0%～30%, Cyclovirobuxine C hydrochloride 0.5%～20%;
Described preparation is injection (comprising injection liquid and freeze-dried preparation) or tablet, capsule, pill, syrup, granule, powder, oral solution.
Useful technique effect of the present invention is: buxine and hydrochloride thereof are made up of cyclovirobuxinum D, cyclobuxine D and Cyclovirobuxine C alkaloid substantially, for by the mixture that directly extracts in the Ramulus Buxi Sinicae, has clear and definite, the stable content of structure, effective quality control can be carried out, the characteristics of determined curative effect can be ensured; The extraction process route is clearer and more definite, easy with respect to cyclovirobuxinum D, cost is lower.Particularly, three kinds of alkaloids also have collaborative cardiovascular disease therapies activity, and buxine and hydrochloride thereof and their corresponding pharmaceutical preparations have the effect that be used for cardiovascular and cerebrovascular diseases more excellent than cyclovirobuxinum D monomer clinically.Main application fields is: coronary heart disease treatment, treating irregular heart pulse, cerebro-vascular diseases treatment.
Buxine is further made its hydrochloride, make its solubility property good, be beneficial to and make various preparations and absorption by human body.
Description of drawings
Fig. 1 analyzes typical figure for buxine HPLC.
Fig. 2 is that buxine hydrochloride HPLC analyzes typical figure.
Fig. 3 is cyclovirobuxinum D reference substance (peak 1).
Fig. 4 measures the cyclovirobuxinum D collection of illustrative plates for HPLC.
Fig. 5 measures buxine hydrochloride collection of illustrative plates for HPLC.
Fig. 6 is the infrared absorption spectrum of buxine.
Fig. 7 is the powder X-ray-diffraction spectra of buxine.
Fig. 8 is the DSC thermogram of buxine.
Fig. 9 is the TG thermogram of buxine.
Figure 10 is the infrared absorption spectrum of buxine hydrochloride.
Figure 11 is the powder X-ray-diffraction spectra of buxine hydrochloride.
Figure 12 is the DSC thermogram of buxine hydrochloride.
Figure 13 is the TG thermogram of buxine hydrochloride.
Embodiment
The invention will be further described below in conjunction with drawings and Examples.
Buxine preparation: the refining alkaloid valid target-buxine that comprises cyclovirobuxinum D, cyclobuxine D and three kinds of main alkaloid compositions of Cyclovirobuxine C of extraction separation from Ramulus Buxi Sinicae.
1.1 buxine preparation
Ramulus Buxi Sinicae is ground into powder, adds 10 times of amount ammonia solns (strong aqua is diluted to 2 times of amount volumes) and soaks 24 hours, and add chloroform and extract three times, the combined chloroform extracting solution, the reclaim under reduced pressure chloroform gets residue.Add sherwood oil dispersion residue, add dilute hydrochloric acid solution (3M) extraction four times, merge dilute hydrochloric acid solution, activated carbon decolorizing adds in the strong aqua and dilute hydrochloric acid solution extremely alkaline (pH12), uses chloroform extraction three times, combined chloroform extracting solution, the most of solvent of pressure reducing and steaming.When having treated that a large amount of solids are separated out, reflux also adds chloroform and just makes dissolving in right amount.Place at cold place, separates out crystallization, filters.With
chloroform recrystallization
2 times, 80 ℃ of drying under reduced pressure, off-white color to faint yellow buxine crystallization.Its solvability sees Table 1.
The solvability of table 1 buxine in common solvent
| Solvent | For examination sampling amount (g) | Complete dissolution solvent consumption (ml) | Solvability |
| Chloroform Glacial acetic acid methanol acetone 0.1mol/L HCl water | 1.0 1.0 1.0 1.0 1.0 1.0 1.0 | 6 9 22 28 90 50 ＞1000 | It is easily molten that easily broad to separate dissolving slightly molten slightly molten almost insoluble |
1.2 form and assay
Chromatographic condition and system suitability test aminopropyl silane group silica gel are weighting agent (Lichrospher-NH2,250mm * 4.6mm, 5 μ m), acetonitrile-0.4% dipotassium hydrogen phosphate solution (70: 30) is a moving phase, flow velocity 1ml/min, 40 ℃ of column temperatures detect wavelength 210nm.
Buxine need testing solution precision takes by weighing the about 5mg of buxine, puts in the 10ml measuring bottle, and it is an amount of to add moving phase, and supersound process makes dissolving, and is diluted to scale, shakes up, promptly.
The measuring method precision is measured need
testing solution
20 μ l, injects liquid chromatograph, and the record color atlas calculates by area normalization method, promptly gets the content of each main alkaloid composition in the trial-product.
Under said system suitability chromatographic condition, but the equal baseline separation of each alkaloid in the buxine bulk drug.Compartment analysis is identified after deliberation, and conclusive evidence this patent method makes the buxine raw material and mainly contains cyclovirobuxinum D, cyclobuxine D and Cyclovirobuxine C.
Liquid phase typical case chromatogram is seen Fig. 1, t among the figure R9.483 the peak is a cyclovirobuxinum D; t R6.862 the peak is cyclobuxine D; t R5.842 the peak is a Cyclovirobuxine C; t R4.917 the peak is without identifying other alkaloid.Show and mainly contain cyclovirobuxinum D (Cyclovirobuxine D), cyclobuxine D (Cyclobuxine D) and three kinds of alkaloid components of Cyclovirobuxine C (Cyclovirobuxine C).
Buxine by weight, content is at (perchloric acid titration in nonaqueous solvent measure, Chinese Pharmacopoeia appendix method) more than 95%.Mainly contain alkaloid components such as cyclovirobuxinum D, cyclobuxine D, Cyclovirobuxine C.
1.3 the content ratio of main component in the buxine
In free alkali, the content ratio of each main alkaloid composition such as following table 2.
Table 2 buxine is formed and content
| Alkaloid component | Content ratio % (wt) |
| Other alkaloid of cyclovirobuxinum D cyclobuxine |
95～60 1.0～30 0.5～20 0～10 |
1.4 the monomeric isolation identification of main alkaloid composition in the buxine
Buxine is to extract refining from Ramulus Buxi Sinicae and alkaloid valid target that get.The present invention utilizes high performance liquid preparative chromatography to carry out the isolation identification of buxine composition after deliberation.Separation condition: preparation of silica gel chromatographic column; Normal hexane-chloroform-methanol-triethylamine (100: 100: 100: 2) moving phase; The ELSD detector.Make cyclovirobuxinum D, cyclobuxine D and Cyclovirobuxine C alkaloid monomer with this condition separation and purification.
The structure of cyclovirobuxinum D identifies that cyclovirobuxinum D is the off-white color crystalline powder, and is easily molten in the chloroform, insoluble in the water.In its EI-MS mass spectrum, the quasi-molecular ions m/z402 in extra best best district is even number, contains the even number nitrogen-atoms in the molecular structure.It is MW402.36 that high resolution mass spectrum obtains its molecular weight, and molecular formula is C 26H 46N 2O, in the KBr compressing tablet IR spectrum at 3414cm -1～3100cm -1, 1147.5cm -1And 1032cm -1Have-NH and-the OH characteristic absorbance.Conclusive evidence is cyclovirobuxinum D.
The structure of cyclobuxine D identifies that cyclobuxine D is the off-white color crystalline powder, and is easily molten in the chloroform, insoluble in the water.In its EI-MS mass spectrum, the quasi-molecular ions m/z386 in extra best best district is even number, contains the even number nitrogen-atoms in the molecular structure.It is MW386.36 that high resolution mass spectrum obtains its molecular weight, and molecular formula is C 25H 42N 2O, comparing decrease in
molecular weight
16 with cyclovirobuxinum D is CH 4The UV spectroscopic analysis shows in the methyl alcohol only end absorption (λ Max=206.8nm), showing does not have conjugated system in the molecular structure, has only heteroatoms amido and hydroxyl or two keys of one of conjugated not.In the KBr compressing tablet IR spectrum at 3414cm -1～3100cm -1, 1147.5cm -1And 1032cm -1Have-NH and-the OH characteristic absorbance; 3092.2cm -1And 3035.1cm -1And 1642.8cm -1And 1605.4cm -1Have with lonely (=C-H) the corresponding absorption of two keys.CDCl 3Middle NMR spectroscopic analysis records δ H4.583/ δ H4.812 alkene hydrogen and the CH2 olefinic carbon (δ of HSQC coupling c100.61) and the relevant alkene quaternary carbon (δ of HMBC c153.55), prove and contain a two key in this alkaloid structure.It is cyclobuxine D (Cyclobuxine D) that this alkaloid is proved conclusively in the multianalysis of NMR spectrum.
The structure of Cyclovirobuxine C identifies that the Cyclovirobuxine C alkaloid is the off-white color crystalline powder.In this product EI-MS mass spectrum, the quasi-molecular ions m/z in extra best best district is 416, is even number, contains the even number nitrogen-atoms in the molecular structure.Obtain its molecular weight MW 416.37 by high resolution mass spectrum, molecular formula is C 27H 48N 2O, comparing molecular weight with cyclovirobuxinum D increases by 14, has promptly increased a methyl substituents on the cyclovirobuxinum D molecular structure.According to its EI-MS spectrum signature cleaved fragment quasi-molecular ions m/e71 (base peak), m/e58 and m/e346 peak, analytical proof compares with cyclovirobuxinum D, and the CH3 of increase is accredited as Cyclovirobuxine C on the secondary amine of 20-position.
1.5 the characteristic light spectrogram of buxine
The typical infrared absorption spectrum of buxine, powder x-diffraction spectra, thermogram as Fig. 6-shown in Figure 9, show that it is the feature crystalline powder respectively.
In the preparation process of buxine, the difference of processing parameter has certain influence to the yield of buxine, but can not have influence on its quality substantially.The parameter of selecting in the present embodiment is a preferred parameter.
Embodiment 2: the buxine hydrochloride
2.1 preparation
Get
buxine
2 grams (pressing method extraction among the embodiment 1), be dissolved in 50 milliliters the chloroform, under stirring state, in chloroformic solution, feed excessive dry hydrogen chloride gas, by every mole of alkaloid (each composition) all with the consumption feeding hydrogen chloride gas of 1～2 mole hcl reaction, promptly generate the buxine hydrochloride precipitation that is insoluble to chloroform, after reaction is finished, leach precipitation, with an amount of washing with alcohol precipitation secondary, 80 ℃ of drying under reduced pressure, with ethanol-chloroform recrystallization, 80 ℃ of drying under reduced pressure promptly get the buxine hydrochloride to constant weight.
Solvability records the solvability of buxine hydrochloride in common solvent and sees Table 3.Compare its solvability in water enlarges markedly with buxine.Solvability in chloroform then significantly reduces.
The solvability of table 3 buxine hydrochloride
| Solvent | For examination sampling amount (g) | Complete dissolution solvent consumption (ml) | Solvability |
| Chloroform methanol Glacial acetic acid 0.1mol/L HCl water | 0.010 0.1 0.1 1.0 1.0 | 200 50 70 20 25 | Almost insoluble slightly soluble slightly soluble dissolving dissolving |
2.2 the buxine hydrochloride is formed
The buxine hydrochloride is that buxine and hydrochloric acid salify make.Buxine and hydrochloric acid salify ratio are: per 1 mole of each composition of alkaloid all with 1～2 mole of hydrogenchloride salify, is mainly dihydrochloride; And contain the absorption and the crystal water of 0～2 molecule, mainly contain 1 molecular crystal water.The content ratio of main alkaloid is identical with buxine in the buxine hydrochloride, and the typical figure that the HPLC of buxine hydrochloride analyzes is seen Fig. 2, and HPLC measures the cyclovirobuxinum D collection of illustrative plates and sees Fig. 3.
The cyclovirobuxinum D reference substance is
peak
1 among Fig. 3.
Buxine hydrochloride-make by buxine and hcl reaction.Buxine hydrochloride content is (the perchloric acid titration in nonaqueous solvent is measured, Chinese Pharmacopoeia appendix method) more than 95%.Mainly contain alkaloid components such as cyclovirobuxinum D, cyclobuxine D, Cyclovirobuxine C.
2.3 the content ratio of main component in the buxine hydrochloride
The content ratio of main alkaloid is identical with buxine in the buxine hydrochloride.In free alkaloid, the content ratio proportional range of each main alkaloid composition sees Table 4
Table 4 buxine hydrochloride is formed and content
| Alkaloid component | Content ratio % |
| Other alkaloid of cyclovirobuxinum D cyclobuxine |
95～60 1.0～30 0.5～20 0～10 |
2.4 the characteristic light spectrogram of buxine hydrochloride
The typical infrared absorption spectrum of buxine hydrochloride, powder x-diffraction spectra, thermogram are seen Figure 10-Figure 13 respectively.Show that it is the feature crystalline powder.
Embodiment 3: the buxine injection
By the injection that buxine and/or its hydrochloride are made, comprise the little pin solution of injection, intravenous drip injection liquid, injectable sterile powder, lyophilize product, etc.Buxine adopts the buxine of preparation among the
embodiment
1, and the buxine hydrochloride adopts the buxine hydrochloride of preparation among the
embodiment
2.
Embodiment 4: the buxine oral preparations
Oral preparations by buxine and/or its hydrochloride are made comprises tablet, capsule, pill, syrup, granule, powder, oral solution.Buxine adopts the buxine of preparation among the
embodiment
1, and the buxine hydrochloride adopts the buxine hydrochloride of preparation among the
embodiment
2.
The pharmacodynamics test example
For beneficial effect of the present invention is described, its test of pesticide effectiveness situation is described further:
1, coronary heart disease treatment
Buxine and hydrochloride thereof and their corresponding pharmaceutical preparations, the diastole coronary artery is relevant with vascular endothelial cell release nitrogen protoxide with function of resisting myocardial ischemia mechanism.Intravenous injection can obviously suppress hemorheology sexual abnormality behind the myocardial ischemia, helps improving myocardial ischemia.To the positive inotropic action of heart mechanism with impel the outer Ca of myocardial cell 2+Ca in interior stream and the myocardial cell 2+Discharge relevant.And the negativity frequency may to be buxine and hydrochloride thereof and their corresponding pharmaceutical preparations cause decreased heart rate to the direct effect of heart, left chamber contractile function strengthens then and discharges relevant with nitrogen protoxide.All effective to symptoms such as the stenocardia of coronary heart disease, uncomfortable in chest, palpitaition, shortnesses of breath.Can significantly reduce the incidence of coronary heart disease and angina pectoris symptom, obviously improve ischemic type electrocardiogram(ECG.Increase aged coronary heart disease cardiac insufficiency person endogenous purple foxglove factor concentration, under comprehensive actions such as coronary artery dilator, blood pressure regulation, improve heart function.Can strengthen myocardial contraction, the expansion peripheral blood vessel alleviates patients with coronary heart disease load diastole, further improves the myocardial blood perfusion, and can strengthen myocardial compliance and Harmony, thereby the patients with coronary heart disease heart function is improved.
2, treating irregular heart pulse
Be applicable to that ventricular premature contraction, tachycardia and chamber quiver etc.And the effect that reduces blood viscosity arranged.Especially shorter to the course of disease, better without other anti-arrhythmic curer or single source premature beat acardia organic lesion person effect.
3, cerebro-vascular diseases treatment
Infringement venereal disease during to acute cerebral ischemia, anoxic becomes to have and improves and provide protection.Cerebral arteriosclerosis, cerebrovascular blood supply insufficiency patient take, and the carotid artery flow amount obviously increases.
One, the monomeric drug effect of buxine hydrochloride and cyclovirobuxinum D relatively
Test materials: cyclovirobuxinum D (white powder crystallization, lot number 2K20526, content＞99.0%).The sodium chloride injection that faces with preceding usefulness 0.9% is mixed with desired concn.HPLC measures the cyclovirobuxinum D collection of illustrative plates and sees Fig. 4.
Buxine hydrochloride (white powder crystallization, lot number 2K2BHCl.The hydrochloride content of cyclovirobuxinum D is that the hydrochloride content of 75% (wt), cyclobuxine D is that the hydrochloride content of 17% (wt), Cyclovirobuxine C is that 5% (wt), other alkaloid component content are 3% (wt)).The sodium chloride injection that faces with preceding usefulness 0.9% is mixed with desired concn.HPLC measures buxine hydrochloride collection of illustrative plates and sees Fig. 5.
1. to the effect of hypoxia tolerance survival time of mouse normal pressure
Experimental technique: get 120 of Kunming mouses, male and female half and half, be divided into 6 groups at random, i.e. control group (giving isometric physiological saline), positive drug group (compound injection of red sage root 10.0g crude drug/kg), cyclovirobuxinum D group (2.0mg/kg), buxine hydrochloride large, medium and small (2.0,1.0,0.5mg/kg).Each group is all by the tail intravenously administrable, and the administration volume is 10ml/kg.Behind the intravenously administrable 10min, mouse is placed the airtight wide-necked bottle of 125ml (built-in sodica calx 20g).Stopping with the mouse mouth breathing is the dead mouse sign, the survival time of record mouse, surpass 60min in 60min.
The buxine hydrochloride sees Table 5 to the provide protection test-results of mouse normal pressure hypoxia tolerance.
Table 5 buxine hydrochloride is to the provide protection of mouse normal pressure hypoxia tolerance
| Group | Dosage (mg/kg) | Number of animals (only) | Breathing hold time (min) |
| The heavy dose of group of dosage group buxine hydrochloride in the control group compound Salviae Miltiorrhizae group cyclovirobuxinum D group buxine hydrochloride small dose group buxine hydrochloride | -10.0g crude drug/kg 2.0 0.5 1.0 2.0 | 20 20 20 20 20 20 | 18.9 ± 7.4 25.2 ± 5.0** 23.6 ± 6.8* ▲ 24.4 ± 9.8 24.9 ± 7.3*, 27.9 ± 5.8** |
| Compare with control group: * P＜0.05, * * P＜0.01; Compare with the heavy dose of group of buxine hydrochloride: ▲: P＜0.05. | |||
Show: buxine hydrochloride height, in two dosage groups (2.0,1.0mg/kg) and cyclovirobuxinum D group (2.0mg/kg) relatively, can obviously prolong the mouse normal pressure hypoxia tolerance survival time.
2. to the effect of Acute Myocardial Ischemia in Rats due to the Pituitrin (Pit)
Experimental technique is got 70 of healthy SD rats, male and female half and half, body weight 180～220g, be divided into 7 groups at random, that is: normal group, model group (above two groups give isometric physiological saline), positive drug group (pannonit 0.5mg/kg), buxine hydrochloride low dose (0.25mg/kg), middle dosage (0.5mg/kg), heavy dose of (1.0mg/kg) 3 dosage groups, cyclovirobuxinum D group (0.5mg/kg).Water 12h is can't help in the rat fasting before the experiment, and dorsal position is got in abdominal injection urethane 1g/kg anesthesia, and it is subcutaneous that needle electrode carefully thrusts four limbs, measures the II lead electrocardiogram, and 30min is stablized in the operation back, and the record normal ECG.Each treated animal is respectively through the tail vein injection administration, and behind the 15min, except that the normal control group, each group is no more than 5s inject time respectively through sublingual vein injection of pituitrin 1.5u/kg.The electrocardiogram(ECG of 15s, 30s, 1,2,3,5,7,10,15min behind the immediate record injection of pituitrin.Observation index mainly is decided to be the variation of T wave height, changes in heart rate.It is baseline that the T wave height is measured with the PR section, and every time point is surveyed 5 successive wave modes, gets its mean value.Record also calculates the changing value (no matter raise or reduce, get the absolute value of variation) of T wave height.For the index that dynamic observes of sequential relationships such as variation of T wave height and heart rate, on each time point, with t check between the changing value work group after the medication.
The buxine hydrochloride sees Table 6 to the influence of rats with myocardial ischemia ECG T wave displacement due to the Pituitrin.
The buxine hydrochloride sees Table 7 to the influence of rats with myocardial ischemia heart rate due to the Pituitrin.
Table 6 buxine hydrochloride is to the influence of rats with myocardial ischemia ECG T wave due to the Pituitrin
(n=10)
| Group | Dosage (mg/k g) | T(mv) | |||||||||
| Before the modeling | After the modeling | ||||||||||
| 15s | 30s | 1min | 2min | 3min | 5min | 7min | 10min | 15min | |||
| The former medicine group of dosage group velocity of variation % hydrochloride heavy dose of group velocity of variation % cyclovirobuxinum D velocity of variation % in the normal group velocity of variation % model group velocity of variation % pannonit group velocity of variation % hydrochloride small dose group velocity of variation % hydrochloride | -- -- 0.5 0.25 0.5 1.0 0.5 | 0.19±0.04 0.19±0.04 0.19±0.07 0.20±0.04 0.20±0.05 0.19±0.05 0.20±0.05 | 0.20±0.04 2.24±2.97 0.54±0.14 ＊＊ 193.11±83.87 ＊＊ 0.25±0.11 ＊＊ 35.14±25.89 ＊＊ 0.50±0.09 147.51±50.75 0.34±0.12 ＊＊ 64.91±38.62 ＊＊ 0.26±0.10 ＊＊ 37.51±29.21 ＊＊ 0.41±0.11 ＊ 110.98±45.84* | 0.20±0.04 3.39±3.01 0.29±0.13 # 60.95±91.7.3 # 0.17±0.12 ＊ -9.99±46.36 ＊ 0.23±0.06 11.59±24.65 0.19±0.12 -9.52±77.53 0.19±0.05 ＊ 6.05±23.90 0.23±0.05 20.22±21.86 | 0.20±0.04 -2.19±13.42 0.20±0.04 2.96±31.49 0.18±0.12 13.43±42.57 0.22±0.08 -8.38±25.81 0.20±0.03 2.25±44.61 0.20±0.05 7.12±36.75 0.24±0.05 3.14±35.41 | 0.20±0.04 -4.03±15.39 0.14±0.05 # -5.71±21.40 0.19±0.06 10.22±39.37 0.18±0.06 ＊ -13.03±24.93 0.18±0.05 -4.63±32.48 0.20±0.05 ＊ 3.34±33.46 0.21±0.06 -0.16±36.37 | 0.20±0.04 2.61±6.58 0.22±0.06 17.53±38.53 # 0.20±0.06 10.05±24.73 0.21±0.07 1.65±28.58 0.21±0.03 10.41±35.68 0.21±0.05 17.21±22.31 0.22±0.08 10.43±24.73 | 0.20±0.04 1.69±4.28 0.24±0.04 # 26.89±21.86 ## 0.23±0.05 33.75±37.04 0.22±0.08 10.60±40.43 0.22±0.03 12.74±35.01 0.23±0.06 26.94±25.00 0.25±0.05 30.69±23.66 | 0.20±0.04 3.09±5.17 0.23±0.05 22.91±24.91 0.21±0.04 22.47±31.03 0.21±0.08 2.73±36.62 0.23±0.04 19.75±44.05 0.22±0.06 17.66±22.62 0.24±0.06 22.57±20.87 | 0.20±0.04 4.67±5.96 0.23±0.06 23.29±35.91 0.23±0.05 28.03±25.45 0.23±0.07 12.77±37.21 0.23±0.04 21.62±45.59 0.23±0.06 23.91±26.92 0.24±0.06 22.15±55.01 | 0.20±0.04 4.40±7.70 0.24±0.05 25.98±29.08 0.22±0.05 22.90±23.31 0.24±0.08 15.03±32.88 0.23±0.04 21.84±45.44 0.23±0.07 26.24±28.44 0.25±0.06 17.00±50.35 |
Compare with model group *P＜0.05, *P＜0.01; Compare with normal group #P＜0.05, ##P＜0.01
Table 7 buxine hydrochloride is to the influence of rats with myocardial ischemia heart rate due to the Pituitrin
(n=10)
| Group | Dosage (mg/kg) | Heart rate (beats/min) | |||||||||
| Before the modeling | After the modeling | ||||||||||
| 15s | 30s | 1min | 2min | 3min | 5min | 7min | 10min | 15min | |||
| The former medicine group of dosage group velocity of variation % hydrochloride heavy dose of group friendshipization rate % cyclovirobuxinum D velocity of variation % in the normal group velocity of variation % model group velocity of variation % pannonit group velocity of variation % hydrochloride small dose group velocity of variation % hydrochloride | -- -- 0.5 0.25 0.5 1.0 0.5 | 395±43.84 417.4±29.35 402.6±71.24 392.4±48.11 389.6±46.56 389.1±30.62 379.9±55.03 | 395±43.84 0±0 346.8±60.98 -16.56±5.31 ** 387.0±70.33 -3.68±8.78 ＊ 351.6±90.78 -10.25±19.78 331.1±41.18 -14.55±10.08 370.2±79.85 -5.24±17.87 328.4±66.42 -13.87±11.16 | 395±43.84 0±0 175.0±62.25 ** -57.89±4.90 ** 286.2±129.22 ＊ -29.29±27.75 ＊ 202.9±42.85 -48.23±10.36 234.5±85.06 -38.47±24.82 * 262.5±101.14 ＊ -32.48±25.51 ＊ 212.0±55.33 -42.82±19.02 | 395±43.84 0±0 196.5±60.55 ** -52.59±5.24 ** 259.9±95.73 -35.51±19.17 ＊ 228.3±44.41 -41.41±11.60 196.2±52.90 -48.58±16.96 240.2±61.88 -38.69±13.50 ＊ 230.0±51.37 -37.78±18.45 | 394±36.80 -0.04±3.12 238.5±53.76 ** -42.74±2.36 ** 285.3±60.66 -27.90±15.52 ＊ 250.3±29.14 -35.70±7.96 215.4±45.47 -43.97±13.95 254.6±56.58 -34.99±12.01 249.3±32.08 -33.04±13.66 | 394±36.85 0.10±3.46 244.2±59.53 ** -41.48±3.00 ** 281.1±70.17 -29.66±13.19 267.6±44.71 -31.37±11.85 236.7±47.92 -38.92±12.42 262.7±54.99 -32.96±11.76 243.3±21.65 -34.84±10.73 | 398±39.24 0.89±1.83 251.1±46.99 ** -39.80±0.16 ** 271.4±54.29 -32.00±9.80 266.4±30.09 -31.63±8.39 256.6±51.26 -34.18±9.59 259.5±48.11 -33.53±10.82 241.5±44.35 -35.46±13.81 | 396±43.70 0.28±1.98 263.0±47.24 ** -36.90±0.36 ** 274.2±44.64 -31.08±8.83 270.6±30.52 -30.71±6.76 268.6±53.15 -30.96±10.97 284.5±49.72 -27.05±11.35 258.0±23.66 -30.93±10.49 | 397±40.8 0.61±1.95 275.4±51.80 ** -33.92±1.94 ** 282.0±53.66 -29.38±9.26 289.8±43.91 -25.59±12.15 260.8±93.87 -33.23±22.10 296.5±48.59 -24.05±10.28 264.0±24.66 -29.12±12.20 | 397±41.78 0.61±3.01 290.7±40.04 ** -36.18±4.80 ** 306.8±74.55 -23.20±14.48 315.0±63.39 -19.42±15.78 297.6±41.30 -23.34±8.66 321.2±55.79 -17.69±12.09 * 301.9±37.98 -19.43±13.07 |
Compare with model group *Compare with normal group P＜0.05 #P＜0.05, ##P＜0.01
The result shows: buxine hydrochloride height, in the variation that can obviously resist the electrocardiogram(ECG T section that Pituitrin causes of two dosage groups, show that the cyclobuxine hydrochloride has the effect of myocardial ischemia due to certain anti-Pit.Behind the rats in normal control group iv Pit, the rat heart rate is obviously slowed down, not recover yet to 10min.The buxine hydrochloride has the tendency of the decreased heart rate due to the alleviation Pit, but not statistically significant (P＞0.05).
3. the effect that the isolated rabbit heart ischemia reperfusion is damaged
36 of experimental technique body weight 2.0-2.4kg rabbit, be divided into 6 groups at random, that is: normal group, model group, positive drug group (compound injection of red sage root, 0.04g crude drug/ml), buxine hydrochloride low dose (0.02mg/ml), middle dosage (0.04mg/ml), heavy dose of (0.08mg/ml) 3 dosage groups.Animal auricular vein injecting heparin shoots execution dead behind the 15min, open chest, heart is taken out rapidly, move in the culture dish of contain cold-penetration stream Rockwell solution, wash out heart blood as far as possible, after the pruning, under liquid level, rapidly aorta is connected on the sleeve pipe of perfusion, and pricks fixing with toe-
in.Feed
95% O immediately 2And 5%CO 2Locke liquid carry out perfusion.Perfusion bottle rise
heart
50～60cm, the perfusate temperature keeps 38 ℃.Three electrodes insert in left and right sides auricle and apex respectively, are connected in two road physiographs, continue rhythm of the heart.After recovering beat of heart and heart rate are steady, stablize 15min, experimentize again.
Modeling is adopted and is stopped irritating the ischemic reperfusion injury: close the aorta sleeve pipe, make heart stop irritating and causing myocardial ischemia, unclamp cartridge clip behind the 45min, recovery nature perfusion.
Administration: medicine is prepared with perfusate, in multiple beginning dabbling drug of irritating, uses the perfusion perfusion behind the 5min.
Buxine hydrochloride height, in two dosage groups damage has provide protection to heart ischemia reperfusion; can improve the coronary flow of Reperfusion Heart; CK and LDH release in the heart behind the inhibition ischemia-reperfusion; the Histological injury of the antagonism cardiac muscle that ischemia-reperfusion caused, prompting buxine hydrochloride has the effect of the reperfusion injury that resists myocardial ischemia.See Table 8 and table 9.
Table 8 buxine hydrochloride is to the influence of LDH in the extrasomatic rabbit heart ischemia-reperfusion myocardial perfusion liquid
(n=6)
| Group | Dosage (mg/ml) | LDH(u/L) | ||||||
| Before stopping irritating | After multiple the filling | |||||||
| 5min | 10min | 15min | 30min | 45min | 60min | |||
| The heavy dose of group of dosage group rate of change % hydrochloride rate of change % in the control group rate of change % model group rate of change % compound Danshen Root group rate of change % hydrochloride small dose group rate of change % hydrochloride | -- -- 40.0 0.02 0.04 0.08 | 15.0 ±4.3 28.8 ±15.8 32.3 ±13.2 14.2 ±10.5 18.7 ±12.8 26.8 ±15.5 | 10.5 ±4.2 -21.2 ±48.5 37.2 ±28.9 87.7 ±163.4 32.2 ±9.0 15.9 ±57.1 7.8 ±7.3 -42.9 ±54.9 26.7 ±55.7 55.5 ±296.2 12.5 ±8.9 -57.2 ±12.8 | 14.2 ±7.3 -0.3 ±48.2 24.5 ±15.0 17.4 ±82.6 34.3 ±11.6 15.3 ±40.0 13.3 ±10.6 1.5 ±51.0 33.2 ±64.8 74.6 ±283.8 21.8 ±14.7 -21.7 ±31.0 | 20.0 ±10.9 41.2 ±78.9 88.2 ±128.6 243.5 ±399.3 59.8 ±95.1 59.1 ±202.6 19.3 ±18.6 47.7 ±80.6 35.5 ±48.4 102.5 ±241.9 23.8 ±17.0 -10.9 ±56.6 | 18.7 ±3.6 ＊ 31.8 ±37.3 ＊ 1382 ±112.9 494.5 ±411.6 63.3 ±42.1 96.3 ±139.0 * 29.3 ±24.0 ＊ 135.3 ±127.3 41.8 ±39.5 150.7 ±179.1 24.5 ±15.2 ＊ 23.9 ±101.3 ＊ | 22.8 ±9.4 ＊＊ 49.4 ±28.4 ＊ 186.5 ±67.2 802.2 ±610.6 76.8 ±63.3 ＊ 156.2 ±228.3 * 31.2 ±19.6 ＊＊ 193.1 ±201.6 ＊ 52.8 ±51.1 ＊＊ 236.9 ±234.7 33.7 ±21.4 ＊＊ 89.9 ±193.4 ＊ | 26.5 ±13.6 ＊＊ 70.9 ±52.1 ＊＊ 217.5 ±89.2 770.4 ±287.3 100.0 ±87.0 ＊ 231.2 ±305.7 ＊ 39.8 ±22.9 ＊＊ 267.3 ±267.9 ＊ 61.8 ±59.8 ＊＊ 273.6 ±231.4 ＊ 41.7 ±24.3 ＊＊ 143.9 ±256.0 ＊＊ |
Compare with model group *P＜0.05, *P＜0.01
Table 9 buxine hydrochloride is to the influence of CK in the extrasomatic rabbit heart ischemia-reperfusion myocardial perfusion liquid
(n=6)
| Group | Dosage (mg/ml) | CK(u/L) | ||||||
| Before stopping irritating | After multiple the filling | |||||||
| 5min | 10min | 15min | 30min | 45min | 60min | |||
| The heavy dose of group of dosage group velocity of variation % hydrochloride velocity of variation % in the control group velocity of variation % model group velocity of variation % compound Salviae Miltiorrhizae group velocity of variation % hydrochloride small dose group velocity of variation % hydrochloride | -- -- 40.0 0.02 0.04 0.08 | 99.5 ±22.0 119.8 ±67.1 65.5 ±60.6 113.7 ±84.6 56.8 ±28.2 133.2 ±87.1 | 101.2 ±19.7 2.7 ±8.7 59.7 ±83.8 -36.4 ±70.1 26.4 ±11.8 -60.4 ±31.7 124.8 ±108.5 12.3 ±65.9 85.7 ±44.8 53.1 ±54.4 140.3 ±103.9 0.4 ±9.8 | 87.8 ±27.8 -13.6 ±16.3 68.0 ±65.9 -31.1 ±57.1 27.8 ±23.9 -36.4 ±35.9 128.0 ±76.7 47.8 ±92.2 62.8 ±33.5 25.3 ±70.6 118.0 ±24.1 11.4 ±46.8 | 92.3 ±34.7 -9.5 ±23.4 256.2 ±273.6 92.6 ±146.3 170.8 ±283.6 314.1 ±462.2 146.2 ±89.9 91.8 ±141.8 63.5 ±25.5 47.0 ±120.3 120.7 ±18.9 19.4 ±61.42 | 115.8 ±42.2 15.8 ±29.4 ＊ 561.2 ±507.3 368.4 ±277.7 86.7 ±75.8 ＊ 39.9 ±85.3 ＊ 229.2 ±134.3 168.7 ±155.6 73.5 ±30.2 ＊ 60.9 ±121.1 ＊ 128.7 ±23.3 23.8 ±55.6 ＊ | 94.0 ±14.2 ＊ -3.8 ±9.7 ＊＊ 817.7 ±638.3 695.0 ±454.4 95.7 ±88.0 ＊ 77.4 ±101.8 ＊＊ 360.3 ±2447 261.6 ±193.1 88.5 ±37.6 ＊ 87.0 ±119.6 ＊ 150.3 ±48.7 ＊ 45.9 ±77.5 ＊＊ | 119.3 ±22.7 ＊ 21.2 ±9.0 ＊＊ 993.8 ±747.5 928.1 ±691.2 231 ±243.6 ＊ 247.1 ±240.7 ＊ 527.2 ±456.4 ＊ 378.3 ±361.9 157.5 ±189.9 ＊ 205.8 ±305.6 ＊ 193.8 ±94.5 ＊ 78.2 ±122.0 ＊ |
Compare with model group *P＜0.05, *P＜0.01
4. to the effect of myocardial infarction due to the anesthetized dog coronary ligation
Test grouping and dosage
Get 35 of hybrid dogs, be divided into 7 groups at random, every group of 5 animals, male and female dual-purpose.
(1) sham operated rats gives isometric(al) physiological saline.(2) model group gives isometric(al) physiological saline.(3) positive drug compound injection of red sage root control group 2g crude drug/kg.(4) buxine hydrochloride small dose group: 0.1mg/kg.(5) dosage group: 0.2mg/kg in the buxine hydrochloride.(6) the heavy dose of group of buxine hydrochloride: 0.4mg/kg.(7) cyclovirobuxinum D group 0.4mg/kg.The used medicine of each treated animal is dissolved in 0.9% sodium chloride injection, and the administration volume is 1ml/kg.T method of inspection and model group, heavy dose of group are carried out statistics relatively with the cyclovirobuxinum D group between the employing group.
Test method is got domesticated dog, and body weight is 7～10Kg, the male and female dual-purpose.With after 3% vetanarcol (30mg/kg) anesthesia, back of the body position is fixed on the operating table, cuts off the hair of neck, chest and left hind inboard by the forelimb small saphenous vein.It is subcutaneous that needle electrode is inserted the dog four limbs, recording ecg (ECG).Separate tracheae and insert trachea cannula.Separate left side femoral artery, femoral vein, separate femoral vein and insert venous cannula, slowly constant speed input physiological saline (about 1ml/min); Separating femoral artery inserts arterial cannulation (being full of the heparin-saline of 500u/ml in the pipe) and connects pressure transducer (TP-400T), and through amplifying (AP-641G) measurement systolic arterial pressure (SAP), auterial diastole pressure (DAP), mean arterial pressure (MAP), calibration sensitivity is 13.33kPa (100mmHg)/cm.The line connection board that connects registering instrument directly triggers heart rate numeration instrument (AT-601G) recorded heart rate (HR) by arterial pressure.Fourth, fifth intercostal is opened chest in the left side, exposes heart, and meets the breathing apparatus.Open pericardium, be sewn in the wall of the chest, make the pocket cradle.Expose right auricle of heart, quiet notes heparin 5～10mg/kg makes heparinization, clamp right auricle of heart with auricular clamp, make a pocket sealing, suture is through in a bit of rubber tubing, line is colluded into rubber tubing with little steel wire, cut an osculum in pocket mouth center, (internal diameter 4～5mm, the mouth of pipe are the oblique angle, and nearly mouth of pipe place is that circle is expanded to insert coronary sinus cannula rapidly, the oblique angle face avoids the mouth of pipe and Dou Bi to be close to, the portion of expanding is for making coronary sinus blood unlikely excessive), with pocket suture tension in the small rubber hose, withstand intubate by small rubber hose, to avoid hemorrhage, rapidly intubate is accurately inserted coronary sinus, ligation right auricle of heart place pocket suture, fixedly intubate.The far-end of this intubate connects a Y-tube to be on the waiting list blood.With descending 1/3 place, a sham operated rats not ligation of threading in No. 0 silk thread ligation ramus descendens anterior arteriae coronariae sinistrae.12 the mapping point seams in position are put the epicardial electrogram electrode near selecting infarct, and selecting a control point away from infarct, carry out mapping with hand-held insulated metal point-like electrode by the mapping dot sequency, through transmitter (JB-642G), amplifier (AB-621G), with eight road physiograph record epicardial electrogram (EECG) (calibrations: 1mm=1mv).Ligation 15min is after systolic arterial pressure (SAP), diastolic pressure (DAP), mean arterial pressure (MAP), heart rate (HR) are directly write down in femoral vein constant flow pump constant speed administration (1ml/min), and traces EECG and arterial pressure (BP) curve in eight road physiographs.Before the record ligation, after ligation 15min and the administration 5,10,15,30,45,60,90, EECG, the SAP of 120min, DAP, MAP, HR.Add up ST field offset ∑-ST of each mapping point EECG, and the ST section is raised 〉=the above N-ST of 2mv.Each group is respectively at before the administration and behind the administration 60min, and left ventricle is got blood and surveyed arterial blood oxygen, and coronary sinus is got blood and surveyed coronary sinus blood oxygen, both oxygen difference reflecting myocardium oxygen consumptions; Each group is respectively at before the ligation, femoral vein is got blood behind ligation 15min, the administration 60min, with kit measurement serum CK, LDH value.After experiment finishes, take out heart immediately, behind physiological saline flush away blood, take by weighing heavy whole-heartedly and ventricular weight, and the ventricle crosscut become 5, and place 37 ℃ of 1%TTC solution 15min that dyes, cut off the non-infarct that each myocardium sheet is colored, undyed infarct cardiac muscle is weighed, obtain infarction size heavily respectively divided by heavy or ventricle whole-heartedly and account for heavy whole-heartedly or account for the heavy percentage of ventricle.
Buxine hydrochloride height, in two dosage groups can slow down that (buxine group effect trend is with physiological saline group comparing difference significantly (P＜0.05) for the HR of anesthetized dog.Buxine hydrochloride group effect is better than cyclovirobuxinum D group (P＜0.05, P＜0.01), table 10), reduce the release of CK, LDH in the serum after the myocardial infarction due to myocardial infarction ∑-ST, N-ST due to the anesthetized dog coronary ligation, the minimizing anesthetized dog coronary ligation, reduce myocardial consumption of oxygen behind the anesthetized dog coronary ligation, reduce myocardial infarction infarction size due to the anesthetized dog coronary ligation, with remarkable (P＜0.05 of physiological saline group comparing difference, P＜0.01), the prompting buxine can obviously alleviate degree of myocardial ischemia.
Table 10 buxine hydrochloride is to the influence of myocardial infarction heart rate (HR) due to the anesthetized dog coronary ligation
(n=5)
| Group | Dosage (mg/kg) | HR (inferior/min) | ||||||||
| Before the administration | After the administration (min) | |||||||||
| 5 | 10 | 15 | 30 | 45 | 60 | 90 | 120 | |||
| Pseudo-operation group velocity of variation % model group velocity of variation % compound Salviae Miltiorrhizae group velocity of variation % small dose group velocity of variation % | - - 2000 0.1 | 172.60 ±40.65 173.60 ±37.06 171.40 ±16.54 178.80 ±16.13 | 171.40 ±35.73 -0.20 ±2.89 158.20 ±41.85 -7.03 ±21.42 169.80 ±17.06 -0.94 ±1.95 179.60 ±17.85 0.42 ±2.96 | 175.00 ±41.36 1.37 ±0.83 177.20 ±38.73 2.06 ±3.16 167.80 ±16.78 -2.13 ±1.63 * 179.80 ±16.38 0.57 ±1.80 | 173.80 ±41.97 0.59 ±1.03 177.20 ±41.50 1.81 ±4.71 168.20 ±15.00 -1.82 ±2.68 178.40 ±15.39 -0.19 ±0.90 | 172.20 ±41.94 -0.35 ±4.87 177.60 ±39.87 2.28 ±5.67 169.60 ±15.79 -1.00 ±3.27 178.80 ±18.67 -0.09 ±2.39 | 172.00 ±41.77 -0.47 ±3.88 176.60 ±40.32 1.67 ±5.80 170.00 ±13.50 -0.66 ±4.30 179.00 ±15.80 0.15 ±2.29 | 172.20 ±38.89 -0.04 ±4.72 179.00 ±37.68 3.36 ±5.33 171.00 ±15.67 -0.18 ±3.89 175.40 ±15.50 -1.88 ±1.51 | 172.20 ±39.16 -0.08 ±3.97 174.80 ±38.67 0.66 ±4.92 168.60 ±21.49 -1.74 ±6.20 176.00 ±17.01 -1.59 ±2.27 | 173.00 ±40.00 0.32 ±4.48 175.00 ±34.68 1.15 ±4.11 167.00 ±21.61 -2.71 ±6.34 169.80 ±20.14 -5.23 ±3.25 |
| The heavy dose of group of middle dosage group velocity of variation % velocity of variation % cyclovirobuxinum D velocity of variation % | 0.2 0.4 0.4 | 165.60 ±31.25 164.40 ±26.35 177.80 ±13.92 | 159.60 ±31.51 -3.69 ±3.22 149.80 ±28.50 -9.12 ±4.82 170.80 ±13.92 -3.95 ±1.34 | 157.80 ±31.13 -4.81 ±1.18 ## 151.20 ±27.80 -8.20 ±4.00 ## 173.40 ±15.16 -2.51 ±2.43 *△ | 158.40 ±33.14 -4.57 ±2.99 * 152.80 ±27.16 -7.19 ±3.43 ## 176.40 ±16.38 -0.86 ±2.71 △ | 161.80 ±33.48 -2.48 ±5.02 157.00 ±30.27 -4.76 ±4.95 175.60 ±14.15 -1.24 ±1.99 | 160.40 ±34.07 -3.42 ±2.39 159.40 ±31.69 -3.37 ±5.27 175.40 ±16.67 -1.41 ±4.01 | 160.00 ±32.35 -3.56 ±2.21 159.80 ±31.29 -3.13 ±4.51 173.40 ±12.40 -2.40 ±3.16 | 158.60 ±32.28 -4.41 ±2.17 158.40 ±33.77 -4.17 ±5.01 175.20 ±12.40 -1.38 ±3.15 | 158.40 ±29.71 -4.33 ±1.45 161.40 ±32.45 -2.19 ±5.32 173.00 ±11.83 -2.53 ±5.18 |
Compare with the physiological saline group *P＜0.05 *P＜0.01
Cyclovirobuxinum D compares with heavy dose of group △P＜0.05
5. to the hemodynamic effect of anesthetized dog
Experimental technique is got 35 dogs, and the male and female dual-purpose is divided into 7 groups at random, and 5 every group, i.e. (1) sham operated rats: give isometric(al) physiological saline; (2) model group: give isometric(al) physiological saline; (3) positive drug compound injection of red sage root control group: 2g crude drug/kg; (4) buxine hydrochloride small dose group: 0.1mg/kg; (5) dosage group: 0.2mg/kg in the buxine hydrochloride; (6) the heavy dose of group of buxine hydrochloride: 0.4mg/kg; (7) cyclovirobuxinum D group: 0.4mg/kg.The used medicine of each treated animal is dissolved in 0.9% sodium chloride injection, and the administration volume is 2ml/kg.
With the subcutaneous cephalic vein anesthesia of 3% vetanarcol 30mg/kg forelimb, back of the body position is fixed on the operating table, cuts off the hair of neck, chest and left hind inboard.Separate tracheae and insert trachea cannula; Separate femoral vein and insert venous cannula, slowly constant speed input physiological saline (about 1ml/min); Separate femoral artery and insert arterial cannulation (being full of the heparin-saline of 500u/ml in the pipe), to measure arteriotony.Under the artificial respiration, open chest, cut off pericardium in the 4th intercostal, be sewn in the wall of the chest, separate root of ascending aorta and ramus descendens anterior arteriae coronariae sinistrae, place the electromagnetic blood flowmeter probe (12mm of suitable internal diameter, 2mm), be connected in and measure cardiac output (CO) and coronary flow (CBF) on the electromagnetic blood flowmeter.Left ventricular cannulation (being full of heparin-saline in the pipe) in left ventricle apex wound inserts left ventricle, is measured left indoor pressure (LVSP), left chamber diastasis pressure (LVEDP), the maximum climbing speed (LVdp/dtmax) of intraventricular pressure; It is subcutaneous that needle electrode is inserted the dog four limbs, recording ecg (ECG).After waiting to stablize, with constant flow pump through femoral vein administration (1ml/min), directly write down systolic arterial pressure (SAP), diastolic pressure (DAP), mean arterial pressure (MAP), heart rate (HR), cardiac output (CO), coronary flow (CBF), and on eight road physiographs, trace ECG, left indoor pressure (LVSP), LVdp/dtmax, LVEDP and arterial pressure (BP) curve.Measure and calculate the BP (SAP of each time period anesthetized dog of administration front and back, DAP, MAP), HR, CO, CBF, LVSP, LVEDP, + dp/dtmax (myocardial contraction parameter),-dp/dtmax (myocardial relaxation parameter), t～dp/dtmax (left chamber begins to be contracted to left indoor pressure climbing speed time to peak), ejection time, SV (stroke output), CI (cardiac index), SI (SI), LVWI (stroke work index), TTI (total oxygen consumption index), TPVR (total peripheral vascular resistance).
Calculation formula:
CI (cardiac index)=CO/ body surface area
SV (stroke output)=CO*1000/HR
SI (SI)=CI*1000/HR
TTI (total oxygen consumption index)=MAP*HR* ejection time
TPVR (total peripheral vascular resistance)=MAP/CO
LVWI (stroke work index)=CI*1.025* (SAP-0.667) * 13.6*0.001
The result: by the result of table 11 as can be seen, can the raise SAP of anesthetized dog of the heavy dose of group of buxine hydrochloride increases stroke output (SV), dog SI (SI), reduces the total oxygen-consumption of anesthetized dog (TTI); Dosage group energy reducing heart rate big or middle increases anesthetized dog coronary flow (CBF, table 7), reduces total peripheral resistance (TPVR), significantly (compares P＜0.05, P＜0.01 with the physiological saline group) with physiological saline group comparing difference.Part-time point effect is better than the cyclovirobuxinum D group.
Table 11 buxine hydrochloride is to the influence of anesthetized dog coronary flow (CBF)
(n=5)
| Group | Dosage (mg/kg) | CBF(ml/min) | ||||||||
| Before the administration | After the administration (min) | |||||||||
| 5 | 10 | 15 | 30 | 45 | 60 | 90 | 120 | |||
| The heavy dose of group of dosage group velocity of variation % velocity of variation % among the physiological saline group velocity of variation % compound Salviae Miltiorrhizae group velocity of variation % small dose group velocity of variation % | - 2000 0.1 0.2 0.4 | 61.14 ±12.04 61.30 ±4.17 59.28 ±3.49 59.56 ±3.11 59.46 ±3.54 | 61.18 ±11.61 0.14 ±1.94 63.96 ±4.65 4.35 ±2.96 60.92 ±4.13 2.74 ±2.28 61.98 ±3.94 4.02 ±1.44 ** 62.74 ±4.66 5.47 | 61.80 ±11.47 1.24 ±2.03 64.20 ±4.82 4.70 ±1.96 60.84 ±4.13 2.60 ±2.44 62.32 ±3.34 4.64 ±1.16 * 62.66 ±4.53 5.35 | 61.32 ±10.94 0.56 ±2.50 64.14 ±4.93 4.61 ±3.02 60.72 ±4.20 2.41 ±2.98 62.00 ±3.35 4.10 ±1.51 * 59.46 ±4.25 5.77 | 61.04 ±11.93 -0.12 ±2.95 63.96 ±4.27 4.38 ±2.73 60.68 ±4.35 2.32 ±2.75 61.66 ±2.19 3.60 ±1.86 * 62.96 ±4.21 5.87 | 61.18 ±11.61 0.20 ±2.55 64.28 ±3.92 4.94 ±3.14 60.94 ±4.44 2.76 ±2.86 61.70 ±2.31 3.67 ±2.37 62.62 ±4.64 5.29 | 60.88 ±11.45 -0.30 ±2.55 64.46 ±3.54 5.27 ±3.44 60.50 ±4.69 2.02 ±3.94 61.16 ±2.46 2.74 ±1.62 63.38 ±4.32 6.58 | 61.52 ±11.71 0.71 ±2.23 63.26 ±5.46 3.12 ±3.60 59.92 ±4.67 1.06 ±4.68 61.08 ±3.39 2.54 ±1.34 63.16 ±4.99 6.19 | 61.42 ±11.19 0.69 ±2.26 63.88 ±4.79 4.20 ±2.75 60.08 ±4.63 1.34 ±4.65 61.10 ±3.45 2.57 ±0.96 63.04 ±4.76 5.99 |
| Cyclovirobuxinum D group velocity of variation % | 0.4 | 59.66 ±8.65 | ±3.22 # 61.10 ±8.62 2.47 ±0.85 | ±3.20 # 61.74 ±9.04 3.46 ±0.51 | ±2.88 # 60.74 ±8.26 1.95 ±2.27 * | ±2.49 ## 60.70 ±8.60 1.80 ±2.28 * | ±3.86 # 60.64 ±8.96 1.62 ±2.16 | ±3.01 ## 60.48 ±8.85 1.36 ±1.76 | ±4.71 # 61.08 ±8.96 2.38 ±3.34 | ±3.84 # 60.80 ±9.23 1.85 ±3.11 |
Compare with the physiological saline group *P＜0.05 *P＜0.01; Compare with the heavy dose group #P＜0.05
6. the comparison of rat plasma pharmacokinetics
The test of the high, medium and low dosage group of buxine hydrochloride (2.0,1.0,0.50mg/kg) rat pharmacokinetics shows the linear dependence that its dosage absorbs.Compare with middle dosage group cyclovirobuxinum D, absorb faster (peak time is shorter, and peak concentration is higher) relative bioavailability higher (187.5 ± 72.9%).Show that its first aid use prospect when clinical treating cardiovascular disease is better.
Above result shows: the buxine hydrochloride has the myocardial ischemia of improvement and heart function effect; Effect is better than cyclovirobuxinum D.
Show by test, the effect that buxine of the present invention and hydrochloride thereof embody in experiment all is better than cyclovirobuxinum D, show between cyclovirobuxinum D in the composition of the present invention, cyclobuxine D and Cyclovirobuxine C and their hydrochloride and have good synergy, though the variation of proportioning has certain influence to effect between three kinds of components, but all be better than the cyclovirobuxinum D monomer, can guarantee the realization of the object of the invention.
Claims (10)
1. buxine, it is characterized in that: be made up of cyclovirobuxinum D, cyclobuxine D and Cyclovirobuxine C substantially, its weight content is respectively cyclovirobuxinum D 60%～95%, cyclobuxine D 1.0%～30%, Cyclovirobuxine C 0.5%～20%.
2. the preparation method of the described buxine of claim 1 is characterized in that: Ramulus Buxi Sinicae is ground into powder, adds 8-12 and doubly measure the ammonia soln immersion, add chloroform and extract, with chloroform extracted solution reclaim under reduced pressure chloroform, get residue; Add sherwood oil and disperse residue, adding dilute hydrochloric acid solution extracts, extracting solution with activated carbon decolorizing after, add in the ammoniacal liquor and dilute hydrochloric acid solution to alkalescence, use chloroform extraction again, with the most of solvent of chloroform extracted solution pressure reducing and steaming, when having treated that a large amount of solids are separated out, reflux also adds chloroform and just makes dissolving in right amount, and place at cold then place, separate out crystallization, filter; With the chloroform recrystallization at least once, drying under reduced pressure gets off-white color to faint yellow buxine crystallization.
3. the preparation method of buxine as claimed in claim 2 is characterized in that: the ammoniacal liquor that described immersion Ramulus Buxi Sinicae powder is used is that strong aqua is diluted to 2 times of ammoniacal liquor behind the volume.
4. the preparation method of buxine as claimed in claim 2, it is characterized in that: described dilute hydrochloric acid is the hydrochloric acid of 3M.
5. the preparation method of buxine as claimed in claim 2, it is characterized in that: described drying under reduced pressure service temperature is 80 ℃.
6. the preparation method of buxine as claimed in claim 2 is characterized in that: described pH value with in the ammoniacal liquor and back solution is 12.
7. buxine hydrochloride, it is characterized in that: described buxine hydrochloride is made up of the hydrochloride of cyclovirobuxinum D, the hydrochloride of cyclobuxine D and the hydrochloride of Cyclovirobuxine C substantially, and its weight content is respectively the hydrochloride 60%～95% of cyclovirobuxinum D, the hydrochloride 1.09%～30% of cyclobuxine D, the hydrochloride 0.5%～20% of Cyclovirobuxine C.
8. the preparation method of the described buxine hydrochloride of claim 7, it is characterized in that: the buxine that will be made up of cyclovirobuxinum D, cyclobuxine D and Cyclovirobuxine C substantially is dissolved in the chloroform, under stirring state, in chloroformic solution, feed excessive dry hydrogen chloride gas, generation is insoluble to the buxine hydrochloride precipitation of chloroform, after reaction is finished, leach precipitation, precipitate with washing with alcohol, through drying under reduced pressure, with ethanol-chloroform recrystallization, drying under reduced pressure obtains the buxine hydrochloride to constant weight again.
9. as the preparation method of claim 8 buxine hydrochloride, it is characterized in that: the mol ratio of described buxine and hydrogenchloride is 1: 1～2.
10. buxine preparation, it is characterized in that: said preparation is made by buxine or its hydrochloride;
Described buxine is made up of cyclovirobuxinum D, cyclobuxine D and Cyclovirobuxine C substantially, and its weight content is respectively cyclovirobuxinum D 60%～95%, cyclobuxine D1.0%～30%, Cyclovirobuxine C 0.5%～20%;
Described buxine hydrochloride is made up of the hydrochloride of cyclovirobuxinum D, the hydrochloride of cyclobuxine D and the hydrochloride of Cyclovirobuxine C substantially, and its weight content is respectively cyclovirobuxinum D hydrochloride 60%～95%, cyclobuxine D hydrochloride 1.0%～30%, Cyclovirobuxine C hydrochloride 0.5%～20%;
Described preparation is injection or tablet, capsule, pill, syrup, granule, powder, oral solution; Described injection comprises injection liquid and freeze-dried preparation.
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| CN112521440B (en) * | 2020-05-25 | 2021-08-20 | 南京经皮科技有限公司 | Method for purifying cyclochrysine D |
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