CN100546589C - The preparation of caterpillar fungus cephalosporin powder anti-arrhythmia effective part and pharmaceutical preparation thereof - Google Patents
The preparation of caterpillar fungus cephalosporin powder anti-arrhythmia effective part and pharmaceutical preparation thereof Download PDFInfo
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Abstract
The invention discloses a kind of the preparation anti-arrhythmia effective part is extracted in industrialization from caterpillar fungus cephalosporin powder new method and pharmaceutical preparation, its method is: with caterpillar fungus cephalosporin powder with soak with ethanol, supersound extraction; Ethanol extraction evenly is suspended in the water fractional extraction after reclaiming solvent; Extract with chloroform or chloroform and methanol mixed solvent elution, obtains antiarrhythmic effective site through silica gel column chromatography.This method is fit to industrialization and prepares in a large number, and the effective site of preparation can be made into various dosage forms, has characteristics such as rapid-action, that day taking dose is little, herbal toxic effect is little.
Description
(1) technical field:
The present invention relates to the method and the pharmaceutical preparation at a kind of extraction from Cordyceps cephalo (C.sinensis Chen.sp.nov) mycopowder, separation, purification antiarrhythmic activity position, this pharmaceutical preparation is used for the treatment of multiple cardiovascular disease such as arrhythmia.
(2) background technology:
Cordyceps is the traditional Chinese crude drug of China.Traditional Chinese medicine theory is thought: Cordyceps sweet in the mouth, warm in nature, the effect of invigorating the lung and the kidney is arranged, and can control chronic cough dyspnea due to deficiency, chronic cough hemoptysis and disease such as the sexual impotence that causes of suffering from a deficiency of the kidney, seminal emission.But because special growing environment and the habit of Cordyceps, make its quantity rareness, scarcity of resources.People separate fungal bacterial strain and carry out large-scale submerged fermentation and cultivate the acquisition Cordyceps powder from Cordyceps.Studies show that: these Cordyceps powder have and Cordyceps similar activity composition and pharmacological action, can replace the Cordyceps medical material to play treatment and tonic effect.At present, having had multiple on the market is that the medicine and the tonic of primary raw material emerges with the Cordyceps powder, and as JINSHUIBAO, mind calming treasured, zhiling capsules etc., these product determined curative effects are sold well, have obtained good social benefit and economic benefit.
Arrhythmia be meant that the change, cardiac impulse of the heart rate and the rhythm and pace of moving things aroused in interest form and (or) conduction abnormalities of impulsion, the category that on the traditional Chinese medical science, belongs to palpitation with fear, palpitation with a distress feeling, clinical manifestation is nervous, cardiopalmus, terrified uneasiness and arrhythmia, with breathe hard, uncomfortable in chest, suppress and breathe heavily and symptom such as dizziness.Reasons such as medicine, myocardial ischemia, electrolyte disturbance, autonomic nervous dysfunction cause that cardiac impulse forms, conduction abnormalities, cause arrhythmia.Arrhythmia is a common disease, and at present, ARR sickness rate does not still have definite statistics.According to interrelated data various arrhythmia sickness rate are compared and to show, wherein the arrhythmia sickness rate is the highest, be 25%～27%, sinus tachycardia is taken second place, and is 20%～22%, sinus bradycardia is 13%～15%, ventricular presystole is 14%～16%, and atrial premature beats is 5%～7%, and atrial fibrillation is 11%～15%, atrioventricular block is 5%～7%, other various arrhythmia about 5%～8%.At present, be used for the treatment of the antiarrhythmic Western medicine and roughly can be generalized into four major types: sodium channel inhibitor, beta receptor blocking agent, over reach current potential time-histories medicine and calcium channel blocker etc.Chang Yong antiarrhythmic drug all has its deficiency clinically: 1. often easily bring out arrhythmia as antiarrhythmic drug, incidence rate is about 3%～13%.2. stronger to the conducting system inhibitory action, the improper or long-term prescription of dosage easily brings out conduction block and heart failure.And compare with Western medicine, there is the stress efficacy low problem of tiring again in arrhythmia Chinese medicine, and its main cause is: 1. to ARR action target spot a little less than the few and effect.2. effect lacks specificity.3. the effective ingredient of compound preparation is indeterminate.Bioavailability was low when 4. middle pharmaceutically active ingredient was oral.These factor affecting the clinical therapeutic efficacy of arrhythmia Chinese medicine.
Isolating Clavicipitaceae fungus Cordyceps cephalo (C.sinensis Chen.sp.nov) obtains caterpillar fungus cephalosporin powder through liquid submerged fermentation from Cordyceps, it contains multiple components such as Cordyceps polysaccharide, protein, aminoacid, mannitol, fat, fatty acid, cordycepin, alkaloid, trace element, material is formed complicated, and effective substance is unclear.
The mind calming health-care capsule is the capsule gained preparation of promptly packing into after the simple drying of caterpillar fungus cephalosporin powder.It has the raising sinus rhythm, improves sinuatrial node, chamber conduction function, improves the effect of cardiac function, is used for the treatment of multiple intractable slow arrhythmia and atrioventricular block.But it is, slow and dosage is big for the arrhythmia onset because the mind calming treasured extracts without modern crafts; And complicated medicine components makes active substance unclear, mechanism of action is not understood, cause lacking effective on-line quality testing and method of quality control in the suitability for industrialized production, thereby influenced quality testing and control, be unfavorable for the further popularization and the utilization of product medical material.
(3) summary of the invention:
Task of the present invention is to adopt modern extraction separation purification technique and compound structure authenticate technology, in conjunction with the modern pharmacological research method, provide a kind of method that industrialization extraction, separation, purification have the antiarrhythmic activity effective site that clear and definite material forms that from caterpillar fungus cephalosporin powder, is fit to;
It is the Chinese medicine preparation that main medicine and other one or more excipient that pharmaceutically allows are made that the present invention also provides this antiarrhythmic activity effective site, is used for the treatment of cardiovascular disease.
Technical scheme of the present invention is:
(1) be the 8%-98% soak with ethanol 12～48 hours of 2～5 times of amounts with the caterpillar fungus cephalosporin powder envelope-bulk to weight ratio, use the 8%-98% ethanol ultrasonic extraction, extract 3 times, each ultrasonic time is 30 minutes, merges 3 times supersound extraction liquid;
(2) reclaim the ethanol extraction that obtains containing effective position behind the alcohol solvent, reuse distilled water supersound extraction 3 times is used the distilled water of 3 times of envelope-bulk to weight ratios at every turn, and ultrasonic time 30 minutes merges 3 times supersound extraction liquid, and the recycle-water solvent obtains water extract; Water extract evenly is suspended in the water, or contains in the alcoholic acid water back organic solvent fractional extraction, water extract and suspendible aqueous solvent or the ratio that contains the volume of alcoholic acid water are 3: 1, and organic solvent is 1: 1 with the ratio of the suspended matter volume that contains effective position;
(3) extract that obtains with chloroform or chloroform and methanol mixed solvent elution, obtains antiarrhythmic effective site through silica gel column chromatography.
Described ethanol extraction evenly is suspended in and contains in the alcoholic acid water ethanol: the ratio of the volume of water is 0.1-1.0: 9.9-9.0.
Described extraction organic solvent is one or more in cyclohexane extraction, chloroform, ethyl acetate, butyl acetate, isopropyl alcohol or the water-saturated n-butanol.
When described eluting was used chloroform and methanol mixed solvent, its weight proportion was 10～1: between 0～1.
The effective site main component that preparation method of the present invention obtains is nucleoside compounds such as adenosine, uridnine and thymidine, can be used as ARR medicine.
This extraction separation purification process step is less, simple to operate, the most of ucleosides composition that is contained in the Cordyceps mycelium powder can be extracted, the antiarrhythmic activity position of final gained, it is mensuration to adopt HPLC to add, and the content that records uridnine in the cephalosporium sinensis filament extract anti-arrhythmia effective part is 30%～50%; And the content of adenosine is 20%～40%.
Said method of the present invention can be used for industrialization and prepare antiarrhythmic activity effective site in a large number, carries out separation and purification by high performance liquid chromatograph, obtains 3 chemical compounds altogether.According to the physicochemical property and the spectral data of chemical compound, determine 3 main compound, be respectively: uridnine, thymidine and adenosine.Its particular chemical is as follows:
Uridnine (uridine) thymidine (thymidine) adenosine (adenosine)
The main compound structure is determined method:
Chemical compound 1: uridnine (uridine), MF:C
9H
12N
2O
6, colourless needle (MeOH), mp168.5-170.0 ℃, MW:244.[ESI-MS]
+ m/z 267[M+Na]
+；245[M+H]
+；113[M-132+H]
+。[ESI-MS]
- m/z 487[2M-H]
-；243[M-H]
-。The molecular weight that can release chemical compound according to above-mentioned mass spectrometric data is 244, contains even number N atom, binding compounds
13CNMR and
1H NMR data, the molecular formula of determining chemical compound is C
9H
12N
2O
6According to chemical compound
13C NMR,
1H NMR and HMQC spectrum has been finished C in the chemical compound and the chemical shift ownership (seeing Table 1) of the H atom that connected.
Table 1
1H and
13C NMR data of compound 1in Pyridine
*
*Recorded on a Bruker AV 400(400MHZ for
1H，100MHz for
13C)NMR spectrometer
Above-mentioned ESI-MS, therefore the uridnine data basically identical in NMR data and the document determines that this chemical compound is uridnine (uridine).
Chemical compound 2: thymidine (thymidine), MF:C
10H
14N
2O
5, yellow powder, mp186-187.5 ℃, MW:242.[ESI-MS]
+m/z 265[M+Na]
+；243[M+H]
+。[ESI-MS]
-m/z 483[2M-H]
-；241[M-H]
-。The molecular weight that can release chemical compound according to above-mentioned mass spectrometric data is 242, contains even number N atom, binding compounds
13C NMR and
1The HNMR data, the molecular formula of determining chemical compound is C
10H
14N
2O
5
According to chemical compound
13C NMR,
1H NMR and HMQC spectrum has been finished C in the chemical compound and the chemical shift ownership (seeing Table 2) of the H atom that connected.
Table 2
1H and
13C NMR data of compound 2in DMSO
*
*Recorded on a Bruker AV 400(400MHZ for
1H，100MHz for
13C)NMR spectrometer
Above-mentioned ESI-MS, therefore the thymidine data basically identical in NMR data and the document determines that this chemical compound is thymidine (thymidine).
Chemical compound 3: adenosine (adenosine), MF:C
10H
13N
5O
4, colourless needle (MeOH), mp 236.8-238 ℃, MW:267.1% anisaldehyde sulphate reagent shows yellow-gray.[ESI-MS]
+m/z 290[M+Na]
+；268[M+H]
+；136[M-132+H]
+。[ESI-MS]
-m/z 533[2M-H]
-；266[M-H]
-；134[M-132-H]
-。The molecular weight that can release chemical compound according to above-mentioned mass spectrometric data is 267, contains odd number N atom, binding compounds
13C NMR and
1H NMR data, the molecular formula of determining chemical compound is C
10H
13N
5O
4
Chemical compound
13C NMR,
1The data of H NMR (Table 3) as shown in the table.
Table 3
1H and
13C NMR data of compound 3 in Pyridine
*
*Recorded on a Bruker AV 400(400MHZ for
1H，100MHz for
13C)NMR spectrometer
This chemical compound and adenosine reference substance are total to thin layer, find that both all have identical Rf value in different development systems, and aforesaid ESI-MS, therefore the adenosine data basically identical in NMR data and the document determines that this chemical compound is adenosine (adenosine).
Press practice of pharmacy, effective site of the present invention can be made the various clinical pharmaceutical dosage form, comprise the dosage form of oral formulations or parenterai administration as the ARR medicine of treatment.Said oral formulations is selected from a kind of in the middle of tablet, capsule, slow releasing tablet, slow releasing capsule, controlled release tablet, controlled release capsule, oral cavity disintegration tablet, drop pill or the soft capsule preparation; The parenterai administration dosage form is selected from a kind of in the middle of cutaneous permeable agent or the injection.
Medicine of the present invention contains 5%～25% effective site and 95%～75% excipient.
Adjuvant in the medicine of the present invention is meant conventional excipient, as starch, PVP, carboxymethyl starch sodium etc.
Cordyceps cephalo of the present invention (C.sinensis Chen.sp.nov) is available from available from pharmaceutical Co. Ltd of Zhejiang Wan Feng enterprise group.
Below the concrete steps of such scheme are elaborated.
1. the preparation of caterpillar fungus cephalosporin powder active effective part
(1) caterpillar fungus cephalosporin powder water extract extractum preparation
Caterpillar fungus cephalosporin powder 1kg doubly measures (V/W) soak with ethanol 12-48 hour with 2-5 after, supersound extraction 3 times, each ultrasonic time is 30 minutes.Merge 3 times supersound extraction liquid, reclaim solvent, promptly obtain the wet extractum of caterpillar fungus cephalosporin powder ethanol extraction.The caterpillar fungus cephalosporin powder reuse distilled water supersound extraction of process ethanol extraction three times, at every turn with 3 times of amounts of distilled water (V/W), ultrasonic 30 minutes.Merge 3 times ultrasonic aqueous extract, reclaim solvent, promptly obtain caterpillar fungus cephalosporin powder water extract extractum.The above-mentioned concentration of alcohol scope that is used to soak and extracts can be between 8%-98%.
(2) extract and separate of caterpillar fungus cephalosporin powder water extract
The water-alcohol extraction extractum of above-mentioned caterpillar fungus cephalosporin powder is dispersed in contains in 1%～10% alcoholic acid distilled water (2000mL), with isopyknic organic solvent (as one or more mixing such as cyclohexane extraction, chloroform, ethyl acetate or water-saturated n-butanols) extraction, extract 3 times.Separating and extracting liquid reclaims solvent, gets the caterpillar fungus cephalosporin powder extract.
(3) caterpillar fungus cephalosporin powder water extract column chromatography for separation
After above-mentioned extract carried out further silica gel column chromatography, eluting promptly gets active effective part of the present invention, and eluting solvent can be selected the mixed solvent of chloroform or chloroform and methanol for use, when selecting chloroform and methanol mixed solvent for use, the ratio of the weight of chloroform and methanol is 10～1: between 1～1.
2. the antiarrhythmic medicine for preparing the caterpillar fungus cephalosporin powder active effective part
The active effective part of the present invention's preparation can directly be made medicine, is applied to patients with arrhythmia by oral, vein, intramuscular injection or other administering mode.Prepare said dosage form on the various pharmaceuticss according to the conventional production method of pharmaceutical field, comprise tablet, capsule, injection, slow releasing tablet, slow releasing capsule, controlled release tablet, controlled release capsule, various cutaneous permeable agent, oral cavity disintegration tablet, drop pill or soft capsule.
The present invention compared with prior art has outstanding characteristics and obvious improvement.
The present invention adopts methods such as extraction, extraction and column chromatography, separation and purification antiarrhythmic activity position from Cordyceps cephalo (C.sinensisChen.sp.nov) mycopowder, and its pharmacologically active is higher than existing Cordyceps powder preparation.
Active effective part among the present invention carries out separation and purification with the preparative high performance liquid chromatography instrument, and ((20 * 250mm) mobile phases are methanol (15/85V/V) for LC-8A preparative high performance liquid chromatography instrument (Japanese Shimadzu Corporation), AQUSAILSS4251 (120) 10 * 250mm and SHIM-PACK PREP-ODS; Flow velocity 1.0mL/min; 30 ℃ of column temperatures), obtain 3 chemical compounds altogether.According to the physicochemical property and the spectral data of chemical compound, determine 3 main compound, be respectively: uridnine, thymidine and adenosine.Mensurationly record that the shared percentage by weight of uridnine is 30%～50% in the cephalosporium sinensis filament extract anti-arrhythmia effective part through adopting HPLC to add, the shared percentage by weight of adenosine is 20%～40%.
The resulting effective site material composition of the present invention is clear, quality standard is controlled.Compare with the mind calming treasured, the effective site main component that the present invention obtains is nucleoside compounds such as adenosine, uridnine and thymidine, this effective site is active high, effectively improve the therapeutical effect of medicine, the drug administration amount is few, the untoward reaction that other impurity component of extract produces human body when avoiding making product again simultaneously.Simultaneously, because active substance understands that product quality is easy to control in the suitability for industrialized production, the reliability height.
Effective site of the present invention can be developed to multiple dosage form, is used for the treatment of multiple cardiovascular disease such as arrhythmia.
The antiarrhythmic drug of the active effective part exploitation of gained of the present invention is used for the treatment of cardiovascular disease, and it is little to have solved existing Cordyceps preparation activity substance content, and dose is big, takes inconvenient problem.And its toxicity of contrast chemical medicine is little, can be made into various dosage forms, as sheet, capsule or injection.
The active effective part of the present invention's preparation shows through in vitro tests: it can resist the myocardial cell antiarrhythmic effect that is brought out by calcium chloride and isoproterenol, and curative effect is suitable with existing anti-arrhythmic.The effective fraction medicine of gained of the present invention, daily dose is few, and the drug action time, early the performance drug action time was long, has obtained beyond thought effect.
The active effective part of the present invention's preparation shows through animal experiment: its scope of application is very wide, has kept the little characteristics of herbal toxic effect.
(4) specific embodiments:
Below, with reference to subsidiary embodiment the present invention being described in more detail, these embodiment are limitation of the present invention anything but.
Embodiment 1: effective site is extracted test 1
Caterpillar fungus cephalosporin powder 1kg is with 8% soak with ethanol of 3 times of amounts (V/W) after 24 hours, supersound extraction 3 times, and each ultrasonic time is 30 minutes.Merge 3 times supersound extraction liquid, reclaim alcohol solvent, promptly obtain caterpillar fungus cephalosporin powder ethanol extraction extractum.Extractum reuse distilled water supersound extraction three times is used distilled water 3000mL at every turn, ultrasonic 30 minutes.Merge 3 times ultrasonic aqueous extract, the recycle-water solvent obtains caterpillar fungus cephalosporin powder water extract extractum.The water-alcohol extraction extractum of above-mentioned caterpillar fungus cephalosporin powder is dispersed in contains in the 10% alcoholic acid distilled water (2000mL), with isopyknic cyclohexane extraction extraction 3 times.Separating and extracting liquid reclaims solvent and gets extract.Above-mentioned extract is carried out further silica gel column chromatography, carries out eluting with chloroform, after promptly get active effective part of the present invention.Be labeled as A1.
HPLC separation, purification and the detection of effective site A1 chemical constituent:
((20 * 250mm) chromatographic columns, eluant methanol are that chromatographically pure, mobile phase are methanol (15/85 for LC-8A preparative high performance liquid chromatography instrument, AQUSAIL SS4251 (120) 10 * 250mm and SHIM-PACK PREP-ODS through the separation and purification of preparative high performance liquid chromatography instrument according to embodiment 1 gained effective site, V/V),), obtain 3 chemical compounds altogether.According to the physicochemical property and the spectral data of chemical compound, determine 3 main compound, be respectively: uridnine, thymidine and adenosine.Through adopting HPLC to add the mensuration content that records uridnine in the cephalosporium sinensis filament extract anti-arrhythmia effective part is 35%; And the content of adenosine is 38.1%, and the content of these two kinds of main components reaches 73.1%.
Embodiment 2: effective site is extracted test 2
Caterpillar fungus cephalosporin powder 1kg is with 98% soak with ethanol of 5 times of amounts (V/W) after 12 hours, supersound extraction 3 times, and each ultrasonic time is 30 minutes.Merge 3 times supersound extraction liquid, reclaim solvent, promptly obtain caterpillar fungus cephalosporin powder ethanol extraction extractum.Extractum reuse distilled water supersound extraction three times is used distilled water 3000mL at every turn, ultrasonic 30 minutes.Merge 3 times ultrasonic aqueous extract, reclaim solvent, obtain caterpillar fungus cephalosporin powder water extract extractum.The water-alcohol extraction extractum of above-mentioned caterpillar fungus cephalosporin powder is dispersed in contains in the 5% alcoholic acid distilled water (2000mL), with isopyknic chloroform extraction 3 times.Separating and extracting liquid reclaims solvent and gets extract.Above-mentioned extract is carried out further silica gel column chromatography, carries out eluting with chloroform and methanol (weight ratio 1: 1), after promptly get active effective part of the present invention.Be labeled as A2.
HPLC separation, purification and the detection of effective site A2 chemical constituent
According to embodiment 1, embodiment 2 gained A2 separate equally uridnine, thymidine and adenosine.Through adopting HPLC to add the mensuration content that records uridnine in the cephalosporium sinensis filament extract anti-arrhythmia effective part is 45.3%; And the content of adenosine is 29.6%, and the content of these two kinds of main components reaches 74.9%.
Embodiment 3 effective sites are extracted test 3
Caterpillar fungus cephalosporin powder 1kg is with 60% soak with ethanol of 2 times of amounts (V/W) after 48 hours, supersound extraction 3 times, and each ultrasonic time is 30 minutes.Merge 3 times supersound extraction liquid, reclaim solvent, promptly obtain caterpillar fungus cephalosporin powder ethanol extraction extractum.Extractum reuse distilled water supersound extraction three times is used distilled water 3000mL at every turn, ultrasonic 30 minutes.Merge 3 times ultrasonic aqueous extract, reclaim solvent, obtain caterpillar fungus cephalosporin powder water extract extractum.The water-alcohol extraction extractum of above-mentioned caterpillar fungus cephalosporin powder is dispersed in contains in the 1% alcoholic acid distilled water (2000mL), with isopyknic isopropyl alcohol extraction 3 times.Separating and extracting liquid reclaims solvent and gets extract.Above-mentioned extract is carried out further silica gel column chromatography, carries out eluting with chloroform and methanol (weight ratio 8: 2), after promptly get active effective part of the present invention.Be labeled as A3.
HPLC separation, purification and the detection of effective site A3 chemical constituent
According to embodiment 1, embodiment 3 gained A3 separate equally uridnine, thymidine and adenosine.Through adopting HPLC to add the mensuration content that records uridnine in the cephalosporium sinensis filament extract anti-arrhythmia effective part is 42.7%; And the content of adenosine is 24.5%, and the content of these two kinds of main components reaches 67.2%.
Embodiment 4: effective site is extracted test 4
Caterpillar fungus cephalosporin powder 1kg is with 80% soak with ethanol of 3.5 times of amounts (V/W) after 30 hours, supersound extraction 3 times, and each ultrasonic time is 30 minutes.Merge 3 times supersound extraction liquid, reclaim solvent, promptly obtain caterpillar fungus cephalosporin powder ethanol extraction extractum.Extractum reuse distilled water supersound extraction three times is used distilled water 3000mL at every turn, ultrasonic 30 minutes.Merge 3 times ultrasonic aqueous extract, reclaim solvent, obtain caterpillar fungus cephalosporin powder water extract extractum.The water-alcohol extraction extractum of above-mentioned caterpillar fungus cephalosporin powder is dispersed in the distilled water (2000mL), with isopyknic water-saturated n-butanol extraction 3 times.Separating and extracting liquid reclaims solvent and gets extract.Above-mentioned extract is carried out further silica gel column chromatography, carries out eluting with chloroform and methanol (weight ratio 9: 1), after promptly get active effective part of the present invention.Be labeled as A4.
HPLC separation, purification and the detection of effective site A4 chemical constituent
According to embodiment 1, embodiment 4 gained A4 separate equally uridnine, thymidine and adenosine.Through adopting HPLC to add the mensuration content that records uridnine in the cephalosporium sinensis filament extract anti-arrhythmia effective part is 48.2%; And the content of adenosine is 30.6%, and the content of these two kinds of main components reaches 78.8%.
Embodiment 5: the active site of the present invention's preparation prepares the anti-arrhythmic capsule preparations
Prescription: the active effective part that the present invention obtains: 2 grams, granular pattern pregelatinized Starch: 18 grams.
Preparation method: get the Cordyceps anti-arrhythmia effective part for preparing among above-mentioned arbitrary embodiment and totally 2 restrain, add filling agent particle type pregelatinized Starch 18 grams, mix homogeneously, incapsulate, altogether 100 of capsules, the gross weight of every capsules is 200mg, effective site content is 20mg.
Usage and consumption: use warm boiled water, each 1,3 times on the one.
Embodiment 6: the active site of the present invention's preparation prepares the arrhythmia tablet
Prescription: the active effective part that the present invention obtains: 3 grams, medical starch: 15 grams, magnesium stearate: 2 grams.
Preparation method: get the Cordyceps anti-arrhythmia effective part for preparing among above-mentioned arbitrary embodiment and totally 3 restrain, add excipient medical starch 15 grams, magnesium stearate lubricant 2 grams, mix homogeneously, make 100 in tablet, the gross weight of every tablet is 200mg, and effective site content is 30mg.
Usage and consumption: use warm boiled water, each 1,3 times on the one.
Embodiment 7: the active site of the present invention's preparation prepares the anti-arrhythmic sustained-release tablet preparation
Prescription: the active effective part that the present invention obtains: 5 grams, hypromellose: 13 grams, polyacrylic resin II:2 gram.
Preparation method: get the Cordyceps anti-arrhythmia effective part for preparing among above-mentioned arbitrary embodiment and totally 5 restrain, add filler hypromellose 13 grams and slow releasing agent polyacrylic resin II 2 grams, mix homogeneously, make 100 of slow releasing tablets, every gross weight is 200mg, and effective site content is 50mg.
Usage and consumption: use warm boiled water, each 1,3 times on the one.
Embodiment 8: the active site of the present invention's preparation prepares the arrhythmia medicine injection
Prescription: the active effective part that the present invention obtains: 2.5 grams, all the other are water for injection, are settled to 500ml.
Preparation method: get the Cordyceps anti-arrhythmia effective part for preparing among above-mentioned arbitrary embodiment totally 2.5 grams add waters for injection and dissolve fully, be settled to 500ml, with filtering with microporous membrane, fill, specification is the 5ml/ bottle, obtain 100 bottles of the total nucleoside injection of Cordyceps, the content of live part is the 25mg/ bottle.
Embodiment 9: the effect experiment of the active effective part of the present invention's preparation
The active effective part of the present invention's preparation has cardiovascular disease effects such as treatment arrhythmia, is confirmed by following effect experiment.
Experiment reagent and material: the active effective part of the present invention's preparation; DMEM/F12 cell culture fluid (Sigma company); Collagenase II type (chemical product is given birth in Shanghai); 5%CaCl
2Injection (going up friendship Jin Zhu pharmaceutcal corporation, Ltd of Hisense product, lot number 030602); Verapamil hydrochloride injection (Shanghai Hefeng Pharmaceutical Co., Ltd.'s product, lot number 020801).1-3 days SD rat (neonatal rat) cardiac muscle cells of former generation of being born; Photoelectricity transducer mount and photoelectricity transducing software system.
1. effective site is tested the myocardial cell arrhythmia vitro inhibition effect that calcium chloride brings out
Method is got the active effective part of above the present invention's preparation respectively, establishes different gradient concentration 0.1,1.0,10.0,50.0,100.0 μ g/ml; The myocardial cell arrhythmia of bringing out with calcium chloride is a model group, makes positive control with verapamil respectively; The beat frequency and the rhythm and pace of moving things of myocardial cell before and after the record administration.Adopt the cell frequency absolute value data of beating to take statistics to learn and handle, the result shows: A1, A2, A3, A4 are when 1.0-10.0 μ g/ml concentration, to the former myocyte's positivity frequency effect effect that improves significantly that nourishes heart of being commissioned to train of the neonatal rat due to the calcium chloride, its action intensity and 10*10
-6μ mol/ml verapamil is suitable.
Effective site is brought out ARR vitro inhibition effect (n=3) to the former myocyte's calcium chloride that nourishes heart of being commissioned to train of neonatal rat
Annotate: group before #:p＜0.05 ##p＜0.01 ###p＜0.001 calcium chloride group vs modeling
*: p＜0.05
*: p＜0.01
* *: p＜0.001 effective site group vs calcium chloride group
The prepared active effective part composition of the present invention as a result is clear and definite, the myocardial cell arrhythmia evidence that calcium chloride is brought out the prepared active effective part of the present invention under low concentration, can obtain the effect suitable with verapamil, possess good arrhythmia effect.
2. effective site the former myocyte's arrhythmia inhibitory action test of nourishing heart of being commissioned to train of neonatal rat that isoproterenol is brought out
Method is got the active effective part of the present invention's preparation respectively, establishes different gradient concentration 0.1,1.0,10.0,50.0,100.0 μ g/ml; The myocardial cell arrhythmia of bringing out with isoproterenol is a model group, makes positive control with Propranolol respectively; The beat frequency and the rhythm and pace of moving things of myocardial cell before and after the record administration.Adopt the cell frequency absolute value data of beating to take statistics to learn and handle, the result shows: A1, A2, A3, A4 are at 10.0 μ g/ml, 50.0 to the former myocyte's positivity frequency effect effect that improves significantly that nourishes heart of being commissioned to train of the neonatal rat due to the isoproterenol, its action intensity is roughly suitable with 0.500 μ g/ml Propranolol during μ g/ml concentration.
Effective site is brought out ARR vitro inhibition effect (n=3) to the former myocyte's isoproterenol that nourishes heart of being commissioned to train of neonatal rat
Annotate:
#: p＜0.05
##P＜0.01
###Group before p＜0.001 isoproterenol group vs modeling
*: p＜0.05
*: p＜0.01
* *: p＜0.001 adds effective position group vs isoproterenol group
The prepared active effective part composition of the present invention as a result is clear and definite, the myocardial cell arrhythmia evidence that isoproterenol is brought out the prepared active effective part of the present invention under low concentration, can obtain the effect suitable with Propranolol, possess good arrhythmia effect.
3. the anxious poison test of effective site
The active effective part that method is got above the present invention's preparation carries out (median lethal dose(LD 50) LD to its acute toxicity
50) measure, experimental result sees the following form:
| Group | Dosage (g/kg) | Log10 dose | Number of animals | Death toll | Mortality rate |
| 1 | 13.710 | 1.1370 | 10 | 9 | 0.9 |
| 2 | 11.790 | 1.0720 | 10 | 8 | 0.8 |
| 3 | 10.140 | 1.0060 | 10 | 8 | 0.8 |
| 4 | 6.720 | 0.9405 | 10 | 4 | 0.4 |
| 5 | 7.499 | 0.875 | 10 | 2 | 0.2 |
| 6 | 6.450 | 0.8096 | 10 | 0 | 0 |
The active effective part of the preparation of the present invention is as a result irritated stomach LD
50Be 9.290g/kg, 95% credibility interval is 8.507-10.14g/kg.
Therefore the anxious malicious test card of effective site understands that the prepared active effective part toxicity of the present invention is low, safe, and good application prospects is arranged.
Claims (9)
1. the preparation method of a caterpillar fungus cephalosporin powder anti-arrhythmia effective part is characterized in that:
(1) with caterpillar fungus cephalosporin powder with the 8%-98% soak with ethanol of 2～5 times of amounts 12～48 hours, use the 8%-98% ethanol ultrasonic extraction then;
(2) reclaim the ethanol extraction that obtains containing effective position behind the solvent, reuse distilled water supersound extraction obtains water extract; Use the organic solvent fractional extraction after evenly being suspended in water extract in the water, water extract is 3: 1 with the ratio of the volume of suspendible aqueous solvent, and organic solvent is 1: 1 with the ratio of the suspended matter volume that contains effective position;
(3) extract that obtains with chloroform or chloroform and methanol mixed solvent elution, obtains antiarrhythmic effective site through silica gel column chromatography.
2. preparation method according to claim 1, it is characterized in that in the step (2), water extract evenly is suspended in contains in the alcoholic acid water back organic solvent fractional extraction, ethanol: the ratio of the volume of water is 0.1～1.0: 9.9～9.0, water extract is 3: 1 with the ratio that the suspendible solvent contains the volume of alcoholic acid water, and organic solvent is 1: 1 with the ratio of the suspended matter volume that contains effective position.
3. preparation method according to claim 1 and 2 is characterized in that: the used organic solvent of fractional extraction is one or more in cyclohexane extraction, chloroform, ethyl acetate, butyl acetate, isopropyl alcohol or the water-saturated n-butanol.
4. preparation method according to claim 1, when it is characterized in that eluting is with chloroform and methanol mixed solvent in the step (3), its weight proportion is 10～1: between 0～1.
5. preparation method according to claim 1 is characterized in that: antiarrhythmic effective site main component is a nucleoside compound: adenosine, uridnine and thymidine can be used as ARR medicine.
6. preparation method according to claim 1 or 5, it is characterized in that: the caterpillar fungus cephalosporin powder anti-arrhythmia effective part that contains the treatment effective dose of method for preparing, add the excipient that one or more pharmaceutically allow, make the dosage form of oral formulations or parenterai administration according to a conventional method, oral formulations is selected from a kind of in the middle of tablet, capsule or the dropping pill formulation; The parenterai administration dosage form is selected from a kind of in the middle of cutaneous permeable agent or the injection.
7. preparation method according to claim 1 or 5, it is characterized in that: the caterpillar fungus cephalosporin powder anti-arrhythmia effective part that contains the treatment effective dose of method for preparing, add the excipient that one or more pharmaceutically allow, make oral formulations according to a conventional method, oral formulations is selected from a kind of in the middle of slow releasing tablet, controlled release tablet, oral cavity disintegration tablet, slow releasing capsule, controlled release capsule or the soft capsule preparation.
8. preparation method according to claim 6 is characterized in that: described pharmaceutical preparation, contain 5%～25% effective site and 95%～75% excipient.
9. preparation method according to claim 7 is characterized in that: described pharmaceutical preparation, contain 5%～25% effective site and 95%～75% excipient.
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