CN100455315C

CN100455315C - Injecting Mailuoning, its preparation and quality control thereof

Info

Publication number: CN100455315C
Application number: CNB200510117482XA
Authority: CN
Inventors: 郭卫芹; 张�育; 齐新英
Original assignee: Shijiazhuang Pharmaceutical Group Ouyi Pharma Co Ltd
Current assignee: Shijiazhuang Pharmaceutical Group Ouyi Pharma Co Ltd
Priority date: 2005-10-31
Filing date: 2005-10-31
Publication date: 2009-01-28
Anticipated expiration: 2025-10-31
Also published as: CN1817365A

Abstract

The present invention provides Mailuoning for injections, a preparation method and a quality control method thereof. The Mailuoning obtained by the preparation method of the present invention has high effective component content and greatly enhances the quality standard of the Mailuoning. Because the contents of chlorogenic acid and cinnamic acid are enhanced, the Mailuoning of the present invention has better curative effects. The present invention simultaneously solves problems such as unstable quality of injections, etc. and has the characteristics of good formability, high dissolution speed, clarity, stability, convenient transportation and operation, portability, etc. The present invention has simple technological steps and is suitable for industrial production.

Description

A kind of injecting Mailuoning and preparation method thereof and method of quality control

Technical field

The present invention relates to field of traditional Chinese medicine pharmacy, particularly a kind of injecting Mailuoning and preparation method thereof and method of quality control.

Technical background

MAILUONING ZHUSHEYE is by one of definite first batch of indispensable Chinese patent medicine of emergency department of national institute of traditional Chinese medicine of State Administration of Traditional Chinese Medicine, ties up to the Qing Dynasty and cures the injection through developing on tame Bao Xiang Ao " New Compilation of Proved Recipes " " SIMIAOYONGAN TANG " basis.This medicine is made by the Chinese medicine of activating blood circulation to dissipate blood stasis such as Herba Dendrobii, Radix Scrophulariae, Radix Achyranthis Bidentatae, Flos Lonicerae, yin nourishing collateral dredging, be the Chinese medicine compound injection, having increases the fiber proenzyme, reduces plasminogen content, suppresses erythrocyte and platelet aggregation, improves viscosity of blood and hypercoagulability, the foundation of promotion collateral circulation, increases the effect of regional blood flow, blood vessel dilating, blood circulation promoting; And have cholinergic effect, smooth muscle spasm be can remove, body's immunity and stress ability adjusted.Its determined curative effect, side effect is little, is mainly used in diseases such as thromboangitis obliterans, venous thrombosis, arteriosclerosis obliterans, cerebral thrombosis and sequela clinically, along with the accumulation of clinical experience, its range of application day by day enlarges, and is used widely clinically.Recent study find wherein the cardiovascular disease resistant active component mainly be chlorogenic acid (Qin Wenjuan etc. Folium Ilicis The Chemical Constituents (II) [J]. Chinese herbal medicine, 1988,19 (11): 6～8), and have hepatic cholagogic effect (Lou Hongxiang etc. the separation of water soluble compound and structure are identified [J] in the Flos Lonicerae. Chinese herbal medicine, 1996,27 (4): 195～199.)。Chlorogenic acid also has inhibitory action to the G-6-Pase that plays a significant role in the regulation and control of balance blood sugar in vivo, help reducing too fast (the Schindler P W of glycogen drainage of noninsulindependent diabetes, Below P, Hemmerle H et al.Identification of two newinhibitors of hepatic glucoe-6-phosphatetranslocase[J] .Diabetologia, 1994,37 (Supp1.1): A134), the mechanism of MAILUONING treatment cardiovascular disease, diabetes and hepatitis, liver cirrhosis is promptly relevant therewith.Chlorogenic acid also has removing and the lipoid peroxidization resistant and the antimutagenesis of free radical in addition.Another active component cinnamic acid is the main component of Radix Scrophulariae, the clinical middle function of increasing leukocyte of finding to have, and zoopery in recent years confirms have leukogenic effect, zoopery also to find to improve peripheral leukocytes and platelet count etc.

Also there be problem and defective in various degree in the production technology of present commercially available MAILUONING ZHUSHEYE, and it mainly comprises: MAILUONING is made aqueous solution, and quality is stable inadequately, be difficult for preserving, regular meeting color and luster occurs and deepens, and crystal is separated out, situations such as composition variation, and transport, store inconvenient; Effective site is extracted incomplete; Preparation quality standard is low, and influence is to the quality inspection and the control of product; As solubilizing agent, and tween 80 can cause hypertension, tachycardia with tween 80, and histamine levels raises and hemolytic side reaction, the insecurity of increase clinical application.

Chinese patent CN1435250A discloses the preparation method of injecting Mailuoning and oral solid formulation, and it adopts methanol, and chloroform is in conjunction with supersound extraction, because methanol, the chloroform boiling point is low, toxicity is big, the preparation that is used for injection is improper, also is unsuitable for commercial production.

Chinese patent CN1555854A, CN1654062A, the disclosed injecting Mailuoning of CN1660314A and preparation method thereof, wherein the active main component chlorogenic acid contents of cardiovascular disease resistant is lower, and effective site is extracted not exclusively, and quality standard is low.

Summary of the invention

The present invention overcomes the deficiencies in the prior art, and a kind of steady quality, injecting Mailuoning that quality standard is high and preparation method thereof are provided.Method of quality control also is provided simultaneously.

Injecting Mailuoning provided by the invention, per 1000 active ingredients of extracting by each 25Kg of Flos Lonicerae, Radix Achyranthis Bidentatae, Radix Scrophulariae, Herba Dendrobii and medically receptible excipient form.

Excipient of the present invention can be in mannitol, inositol, lactose, glucosan, the sodium chloride one or more, consumption is that 0.2-0.4g/ props up.

Cinnamic acid content in the injecting Mailuoning of the present invention is no less than that 3.6mg/ props up, chlorogenic acid content is no less than 150mg/ and props up.

Cinnamic acid content among the present invention can reach that 3.6-5mg/ props up, chlorogenic acid content can reach 150-180mg/ and prop up.

The preparation method of injecting Mailuoning provided by the invention is to make by following steps:

(1) makes 1000 by Chinese medicine Flos Lonicerae 25kg, Radix Scrophulariae 25kg, Radix Achyranthis Bidentatae 25kg, Herba Dendrobii 25kg;

(2) get above four Chinese medicine material, add 50%～70% ethanol 70～80 ℃ of following reflux, extract, 1～3 time, each 6～10 times of amounts, each 1～2 hour, merge ethanol extract, decompression recycling ethanol is not continuously waved to there not being the alcohol flavor with jacket steam;

(3) add 1-2 and doubly measure water for injection, fully stir, insulation was 30-60 minute after jacket steam was heated to 90～95 ℃, cold preservation 12～24 hours, and with the centrifugal 15-30 of the speed of 5000rpm minute, it was standby to get supernatant;

(4) supernatant is transferred pH to 2.0～4.0 with hydrochloric acid, with the ethyl acetate extraction of 1～2 times of amount 4～8 times, combining extraction liquid, behind the reclaim under reduced pressure ethyl acetate, waves to no ethyl acetate with jacket steam and to distinguish the flavor of;

(5) add injection water 10-15L, fully stir the back and filter, filtrate is concentrated into an amount of volume, drying, intermediate;

(6) get above-mentioned intermediate, add excipient, add water make it the dissolving after, transfer pH to 6.0～8.0 with 15-20%NaOH solution, add 1-2 ‰ active carbon, stirred 15-30 minute, filter, it is 5000ml～8000ml that filtrate is suitably replenished water for injection to total amount, by the microporous filter membrane filtrate for later use of 0.22 μ m;

(7) filtrate is propped up packing with 5～8ml/, half moulding plug, and the sabot inlet carries out lyophilizing by freeze-drying curve, and vacuum is put in tamponade after the lyophilizing, and outlet rolls aluminium lid, and check is labelled, packing, promptly.Drying means of the present invention can be a spray drying, vacuum drying a kind of.

The preferred version of preparation method of the present invention is:

(2) get above four Chinese medicine material, add 60% ethanol 70 ℃ of following reflux, extract, 2 times, each 8 times of amounts, each 1 hour, merge ethanol extract, decompression recycling ethanol is not continuously waved to there not being the alcohol flavor with jacket steam;

(3) add 1 times of amount water for injection, fully stir, insulation was 30 minutes after jacket steam was heated to 95 ℃, cold preservation 24 hours, and with the speed of 5000rpm centrifugal 15 minutes, it was standby to get supernatant;

(4) supernatant is transferred pH to 3.0 with hydrochloric acid, with the ethyl acetate extraction of 2 times of amounts 8 times, combining extraction liquid, behind the reclaim under reduced pressure ethyl acetate, waves to no ethyl acetate with jacket steam and to distinguish the flavor of;

(5) add injection water 10L, fully stir the back and filter, filtrate is concentrated into an amount of volume, drying, intermediate;

(6) get above-mentioned intermediate, add excipient, after adding water and making it dissolving, transfer pH to 7.0 with 20%NaOH solution, add 1 ‰ active carbons, stirred 15 minutes, filter, it is 7000ml that filtrate is suitably replenished water for injection to total amount, by the microporous filter membrane filtrate for later use of 0.22 μ m;

(7) filtrate is propped up packing with 7ml/, half moulding plug, and the sabot inlet carries out lyophilizing by freeze-drying curve, and vacuum is put in tamponade after the lyophilizing, and outlet rolls aluminium lid, and check is labelled, packing, promptly.

Excipient used in the present invention is mannitol preferably, and addition preferably 0.3g/ is propped up.

The method of quality control of injecting Mailuoning provided by the invention is as follows:

One, differentiate:

A. the thin layer chromatography of Flos Lonicerae is differentiated;

Get the chlorogenic acid reference substance, add methanol and make the solution that every 1ml contains 1mg, in contrast product solution.Extracting honeysuckle medicinal powder 5g adds methanol 50ml, and supersound extraction 30min filters, and filtrate evaporate to dryness, residue add dissolve with methanol and be settled to 2ml, in contrast medical material solution.Get 1 bottle of this product, add methanol 50ml, supersound extraction 30min, centrifugal, the supernatant evaporate to dryness, residue is with dissolve with methanol and be settled to 10ml, as need testing solution.Other gets Radix Achyranthis Bidentatae, Herba Dendrobii, Radix Scrophulariae medical material, prepares finished product according to manufacturing technique requirent, and the back makes negative preparation need testing solution according to the preparation method of above-mentioned need testing solution.Test according to thin layer chromatography, draw reference substance solution 5 μ l, medical material contrast solution 3 μ l, need testing solution 3 μ l, negative preparation need testing solution 3 μ l, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose, be 20 with the volumetric usage ratio; The upper solution of butyl acetate-formic acid of 5: 5-water is developing solvent, launches, and takes out, and dries, and puts that wavelength 365nm inspects at the place under the ultra-violet lamp.In the test sample chromatograph, with reference substance and the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color;

B. the thin layer chromatography of Radix Scrophulariae is differentiated;

Get Radix Scrophulariae medicinal powder 5g, add water saturated n-butyl alcohol 50ml, supersound extraction 30min takes out, and with the centrifugal 10min of the speed of 7000r/min, supernatant evaporate to dryness, residue add dissolve with methanol and be settled to 2ml, in contrast medical material solution.Get one bottle of this product, be dissolved in water and be settled to 10ml, with water saturated n-butanol extraction three times, consumption is respectively 20ml, 20ml, 15ml, and extract merges, and water-bath volatilizes, and residue adds dissolve with methanol and is settled to 10ml, as need testing solution.Negative preparation need testing solution: get Radix Achyranthis Bidentatae, Herba Dendrobii, Chinese medicine honeysuckle, prepare finished product according to manufacturing technique requirent, the back makes negative preparation need testing solution according to the preparation method of need testing solution.Test according to thin layer chromatography, draw control medicinal material solution 5ul, need testing solution 5 μ l, negative preparation need testing solution 5 μ l, put respectively in same be on the silica gel g thin-layer plate of binding agent with the sodium carboxymethyl cellulose, with the volumetric usage ratio is 2-7: 1 chloroform: methanol solution is developing solvent, launch, take out, dry, spray is inspected under the daylight with 5% vanillin sulphuric acid test solution.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color;

C. the thin layer chromatography of Radix Achyranthis Bidentatae is differentiated;

It is an amount of to even up pier fruit acid reference substance, adds methanol and makes the solution that every 1ml contains 1mg, in contrast product solution;

Get Radix Achyranthis Bidentatae medicinal powder 10g, add ethanol or methanol 50ml, reflux 90min, filter, filtrate adds hydrochloric acid 4ml, reflux 2 hours, be concentrated into about 10ml, add water 20ml, extract three times down for 60～90 ℃ with petroleum ether, consumption is respectively 40ml, 40ml, 20ml, combining extraction liquid, water bath method, residue add dissolve with methanol and are settled to 2ml, in contrast medical material solution.Get 1 bottle of this product, add ethanol or methanol 50ml, reflux 90min, filter, filtrate adds hydrochloric acid 4ml, reflux 2 hours, be concentrated into about 10ml, add water 20ml, extract three times down for 60～90 ℃ with petroleum ether, consumption is respectively 40ml, 40ml, 20ml, combining extraction liquid, water bath method, residue add dissolve with methanol and are settled to 2ml, as need testing solution.Other gets Radix Scrophulariae, Herba Dendrobii, Chinese medicine honeysuckle, prepares finished product according to manufacturing technique requirent, and the back makes negative preparation need testing solution according to the preparation method of need testing solution.Test according to thin layer chromatography, draw reference substance solution 3 μ l, control medicinal material solution 6 μ l, need testing solution 3 μ l, negative preparation need testing solution 3 μ l, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with the volumetric usage ratio is 10-20: 1 chloroform: methanol solution is developing solvent, launches, and takes out, dry, spray is with 10% ethanol solution of sulfuric acid, and it is clear to be heated to the speckle colour developing at 105 ℃, puts that wavelength 365nm inspects at the place under the ultra-violet lamp.In the test sample chromatograph, with reference substance and the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color respectively;

D. the thin layer chromatography of Herba Dendrobii is differentiated;

It is an amount of to get the escoparone reference substance, and chlorination is copied into the solution that every 1ml contains 0.3mg, in contrast product solution.Get Herba Dendrobii medicinal powder 5g, moistening with ammonia, soak close plug and place 30min, add chloroform 100ml, reflux 2 hours is taken out, and filters, and the filtrate evaporate to dryness adds the chloroform dissolving and is settled to 2ml, medical material solution in contrast.Get 1 bottle of this product, powder is moistening with ammonia, soaks close plug and places 30min, adds chloroform 100ml, and reflux 2 hours is taken out, and filters, and the filtrate evaporate to dryness adds the chloroform dissolving and is settled to 10ml, as need testing solution.Other gets Radix Achyranthis Bidentatae, Flos Lonicerae, Radix Scrophulariae medical material, prepares finished product according to manufacturing technique requirent, and the back makes negative preparation need testing solution according to the preparation method of above-mentioned need testing solution.Test according to thin layer chromatography, draw reference substance solution 2 μ l, control medicinal material solution 3 μ l, need testing solution 3 μ l, negative preparation need testing solution 3 μ l, put respectively in same be on the silica gel g thin-layer plate of binding agent with the Sodium Tvlose, with the volume ratio is 0.5-4: 1 petroleum ether: ethyl acetate is developing solvent, launch, take out, dry, put that wavelength 365nm inspects at the place under the ultra-violet lamp.In the test sample chromatograph, with reference substance and the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color;

Two, assay:

A. cinnamic acid is according to high effective liquid chromatography for measuring;

Chromatographic condition and system suitability test octadecylsilane chemically bonded silica are filler; Volume ratio is that 54: 46 methanol-0.1% phosphoric acid solution is a mobile phase; The detection wavelength is 278nm.Theoretical cam curve is calculated by the cinnamic acid peak should be not less than 1500;

It is an amount of that the preparation precision of reference substance solution takes by weighing the cinnamic acid reference substance, and the methanol-water solvent that adds volume ratio and be 54: 46 is made the solution that every 1ml contains 260 μ g, promptly;

This product content under the content uniformity item is got in the preparation of need testing solution, mixing, get 0.2g, the accurate title, decide, and puts in the 50ml volumetric flask, add volume ratio and be 54: 46 methanol-water dissolving and be diluted to scale, shake up, therefrom accurate again the absorption in 5ml to the 10ml volumetric flask, with above-mentioned solvent dilution to scale, shake up, promptly;

Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly;

Every of this product contains cinnamic acid C9H8O2 must not be less than 3.60mg;

B. chlorogenic acid is according to high effective liquid chromatography for measuring;

Chromatographic condition and system suitability test octadecylsilane chemically bonded silica are filler, are that acetonitrile-0.1% phosphoric acid solution of 5-35: 95-65 is a mobile phase with the volume ratio, and the detection wavelength is 327nm.Number of theoretical plate is not less than 1500 by the chlorogenic acid peak;

It is an amount of that the chlorogenic acid reference substance is got in the preparation of reference substance solution, accurate claims surely, puts in the brown volumetric flask, and the acetonitrile-water solvent that adds volume ratio and be 5-35: 95-65 is made the solution that contains 80 μ g among every 1ml;

This product content under the content uniformity item is got in the preparation of need testing solution, mixing, get 0.2g, the accurate title, decide, and puts in the brown measuring bottle of 50ml, to add volume ratio be the acetonitrile-water dissolution with solvents of 5-35: 95-65 and be diluted to scale, shake up, therefrom accurate again the absorption in the brown measuring bottle of 1ml to 25ml, with above-mentioned solvent dilution to scale, shake up, promptly;

Every of this product contains chlorogenic acid in C16H18O9, must not be less than 150.0mg;

Final definite this product is the quantitative target of this product quality with cinnamic acid and chlorogenic acid;

Three, finger printing:

According to high performance liquid chromatography, and in conjunction with the requirement of Chinese medicine chromatographic fingerprinting experimentation technical manual;

Chromatographic condition and system suitability test;

With octadecylsilane chemically bonded silica is filler, is mobile phase with acetonitrile-0.1% aqueous formic acid, and according to the form below carries out gradient elution, and the detection wavelength is 254nm, 35 ℃ of column temperatures.Number of theoretical plate is not less than 3000 by the chlorogenic acid peak;

The gradient elution program is:

It is an amount of that the chlorogenic acid reference substance is got in the preparation of object of reference, with dissolve with methanol and dilution, makes every 1ml and contain the solution of 20 μ g as object of reference solution; It is an amount of to get the cinnamic acid reference substance, with dissolve with methanol and dilution, makes every 1ml and contains the solution of 15 μ g as object of reference solution;

One of this product is got in the preparation of test sample, adds the redistilled water dissolving, puts in the 10ml measuring bottle, and thin up is to scale, shake up, the accurate absorption gone up in liquid 0.2ml to the 25ml measuring bottle, adds redistilled water and is diluted to scale, filters with 0.45 μ m filter membrane, discard filtrate just, get subsequent filtrate, promptly;

The accurate need testing solution 20 μ l that draw of algoscopy inject chromatograph of liquid, and be 70min writing time, gradient equilibration time 10min;

The test sample collection of illustrative plates should to contrast collection of illustrative plates consistent with injecting Mailuoning, and A version in 2004 is calculated through chromatographic fingerprints of Chinese materia medica similarity evaluation system, and similarity should be greater than 0.9;

The contrast collection of illustrative plates of injecting Mailuoning is seen Fig. 2.

Mobile phase can be plurality of color spectra system and gradient elution programs such as methanol-water, acetonitrile-water, methanol-0.05～0.5% phosphoric acid solution, acetonitrile-0.05～0.5% phosphoric acid solution and acetonitrile-0.01～0.2% formic acid solution in the fingerprint spectrum method.

The pH value of injecting Mailuoning of the present invention, insoluble granule, clarity, heavy metal, arsenic salt, protein, tannin, oxalates, potassium ion, resin, moisture, hemolytic test, aseptic, residue on ignition inspection all meet the pertinent regulations under " specification requirement of study of tcm new drug " and " Chinese Pharmacopoeia 2000 one one " injection item.

The present invention determines that by great deal of experimental the technical specification of injecting Mailuoning of the present invention is as follows:

Adopt technology of the present invention to extract, can obtain high-load chlorogenic acid and cinnamic acid, active constituent content obviously improves, and the quality standard of product also improves greatly simultaneously.Because the active component of MAILUONING treatment cardiovascular disease, diabetes and hepatitis, liver cirrhosis mainly is a chlorogenic acid.Find to have function of increasing leukocyte during another active component cinnamic acid is clinical, also find to improve peripheral leukocytes and platelet count etc.So injecting Mailuoning of the present invention has better therapeutic because of the content that has improved chlorogenic acid and cinnamic acid greatly.

The present invention has solved problems such as aqueous injection quality instability simultaneously, has good moldability, instant, clear and bright, stable, accumulating, carries and characteristics such as easy to use.

Processing step of the present invention is simple, is suitable for commercial production.

Description of drawings

The typical finger printing of Fig. 1 injecting Mailuoning;

The gradient elution collection of illustrative plates of Fig. 2 reference substance (chlorogenic acid);

The gradient elution collection of illustrative plates of Fig. 3 reference substance (cinnamic acid);

The gradient elution collection of illustrative plates of Fig. 4 blank solvent;

2 hours collection of illustrative plates of Fig. 5 injecting Mailuoning.

The specific embodiment

Below experiment will further specify the present invention.

Embodiment 1:

Get each 25kg of recipe quantity four Chinese medicine material, add 8 times of amount 60% alcohol reflux 3 times, each 1.5 hours, merge ethanol extract, decompression recycling ethanol, continuous not waving to there being alcohol with jacket steam distinguished the flavor of, add 1 times of water gaging, fully stir, insulation was 30 minutes after jacket steam was heated to 95 ℃, cold preservation 24 hours, centrifugal (5000rpm, 15 minutes) are got supernatant and are transferred pH to 2.0 with hydrochloric acid, with the ethyl acetate extraction of 2 times of amounts 6 times, combining extraction liquid behind the reclaim under reduced pressure ethyl acetate, is waved to no ethyl acetate flavor with jacket steam, add injection water 10L, fully stir the back and filter, filtrate is concentrated into an amount of volume, 60 ℃ of vacuum dryings, get intermediate, detect intermediate content.Get above-mentioned intermediate, add mannitol 300g, after adding water and making it dissolving in right amount, transfer pH to 7.0 with 20%NaOH solution, add 1 ‰ active carbons, stirred 15 minutes, filter, it is 7000ml that filtrate is suitably replenished water for injection to total amount, by the microporous filter membrane of 0.22 μ m, filtrate is propped up packing with 7ml/, half moulding plug, sabot inlet, carry out lyophilizing by freeze-drying curve, vacuum is put in tamponade after the lyophilizing, outlet rolls aluminium lid, check, label, packing, promptly.

Embodiment 2:

Get each 25kg of recipe quantity four Chinese medicine material, add 6 times of amount 60% alcohol reflux 3 times, each 2 hours, merge ethanol extract, decompression recycling ethanol, continuous not waving to there being alcohol with jacket steam distinguished the flavor of, add 1 times of water gaging, fully stir, insulation was 30 minutes after jacket steam was heated to 95 ℃, cold preservation 24 hours, centrifugal (5000rpm, 15 minutes) are got supernatant and are transferred pH to 3.0 with hydrochloric acid, with the ethyl acetate extraction of 2 times of amounts 6 times, combining extraction liquid behind the reclaim under reduced pressure ethyl acetate, is waved to no ethyl acetate flavor with jacket steam, add injection water 10L, fully stir the back and filter, filtrate is concentrated into an amount of volume, 80 ℃ of vacuum dryings, get intermediate, detect intermediate content.Get above-mentioned intermediate, add mannitol 300g, after adding water and making it dissolving in right amount, transfer pH to 7.0 with 20%NaOH solution, add 1 ‰ active carbons, stirred 15 minutes, filter, it is 5000ml that filtrate is suitably replenished water for injection to total amount, by the microporous filter membrane of 0.22 μ m, filtrate is propped up packing with 5ml/, half moulding plug, sabot inlet, carry out lyophilizing by freeze-drying curve, vacuum is put in tamponade after the lyophilizing, outlet rolls aluminium lid, check, label, packing, promptly.

Embodiment 3:

(2) get above four Chinese medicine material, add 50% ethanol 70 ℃ of following reflux, extract, 1 time, each 6 times of amounts, each 1 hour, merge ethanol extract, decompression recycling ethanol is not continuously waved to there not being the alcohol flavor with jacket steam;

(3) add 1 times of amount water for injection, fully stir, insulation was 30 minutes after jacket steam was heated to 90 ℃, cold preservation 12 hours, and with the speed of 5000rpm centrifugal 15 minutes, it was standby to get supernatant;

(4) supernatant is transferred pH to 2.0 with hydrochloric acid, with the ethyl acetate extraction of 1 times of amount 4 times, combining extraction liquid, behind the reclaim under reduced pressure ethyl acetate, waves to no ethyl acetate with jacket steam and to distinguish the flavor of;

(5) add injection water 10L, fully stir the back and filter, filtrate is concentrated into an amount of volume, spray drying, intermediate;

(6) get above-mentioned intermediate, add excipient inositol 200g, after adding water and making it dissolving, transfer pH to 6.0 with 15%NaOH solution, add 1 ‰ active carbons, stirred 15 minutes, filter, it is 5000ml that filtrate is suitably replenished water for injection to total amount, by the microporous filter membrane filtrate for later use of 0.22 μ m;

(7) filtrate is propped up packing with 5ml/, half moulding plug, and the sabot inlet carries out lyophilizing by freeze-drying curve, and vacuum is put in tamponade after the lyophilizing, and outlet rolls aluminium lid, and check is labelled, packing, promptly.

Embodiment 4:

(2) get above four Chinese medicine material, add 50%～70% ethanol 70～80 ℃ of following reflux, extract, 3 times, each 10 times of amounts, each 2 hours, merge ethanol extract, decompression recycling ethanol is not continuously waved to there not being the alcohol flavor with jacket steam;

(3) add 1-2 and doubly measure water for injection, fully stir, insulation was 60 minutes after jacket steam was heated to 95 ℃, cold preservation 24 hours, and with the speed of 5000rpm centrifugal 30 minutes, it was standby to get supernatant;

(4) supernatant is transferred pH to 4.0 with hydrochloric acid, with the ethyl acetate extraction of 2 times of amounts 8 times, combining extraction liquid, behind the reclaim under reduced pressure ethyl acetate, waves to no ethyl acetate with jacket steam and to distinguish the flavor of;

(5) add injection water 15L, fully stir the back and filter, filtrate is concentrated into an amount of volume, vacuum drying, intermediate;

(6) get above-mentioned intermediate, add excipient sodium chloride 300g, after adding water and making it dissolving, transfer pH to 8.0 with 20%NaOH solution, add 2 ‰ active carbons, stirred 30 minutes, filter, it is 8000ml that filtrate is suitably replenished water for injection to total amount, by the microporous filter membrane filtrate for later use of 0.22 μ m;

(7) filtrate is propped up packing with 8ml/, half moulding plug, and the sabot inlet carries out lyophilizing by freeze-drying curve, and vacuum is put in tamponade after the lyophilizing, and outlet rolls aluminium lid, and check is labelled, packing, promptly.

Embodiment 5:

(5) add injection water 10L, fully stir the back and filter, filtrate is concentrated into an amount of volume, vacuum drying, intermediate;

(6) get above-mentioned intermediate, add excipient lactose 100g, Dextran 200 g, add water make it the dissolving after, transfer pH to 7.0 with 20%NaOH solution, add 1 ‰ active carbons, stirred 15 minutes, filter, it is 7000ml that filtrate is suitably replenished water for injection to total amount, by the microporous filter membrane filtrate for later use of 0.22 μ m;

Comparative example 1

This example is that alcohol extraction and traditional extraction process by water compare.

Alcohol extraction and traditional extraction process by water are compared, extracting method is intended electing as decocting with water extraction, 70% ethanol, 60% ethanol and four kinds of methods of alcohol reflux of 50%, extracting yield with main active ingredient cinnamic acid in main active ingredient chlorogenic acid and the Radix Scrophulariae in the Flos Lonicerae is main evaluation index, these four kinds of methods are compared extraction solvent and extracting method that screening is suitable.

1, the preparation of Different Extraction Method sample liquid

(1) decocting boils: take by weighing Flos Lonicerae, Radix Scrophulariae, Radix Achyranthis Bidentatae, each 10g of Herba Dendrobii pharmaceutical decocting piece, add the water of 10 times of amounts, decoct 2 times, each 1.5h filters, and merging filtrate concentrates standardize solution in the 100ml volumetric flask.

(2) 50% alcohol refluxs: take by weighing Flos Lonicerae, Radix Scrophulariae, Radix Achyranthis Bidentatae, each 10g of Herba Dendrobii pharmaceutical decocting piece, add the ethanol of 8 times of amounts 50%, reflux 3 times, each 1.5 hours, merge alcohol reflux liquid, filter, concentrate standardize solution in the 100ml volumetric flask.

(3) 60% alcohol refluxs: take by weighing Flos Lonicerae, Radix Scrophulariae, Radix Achyranthis Bidentatae, each 10g of Herba Dendrobii pharmaceutical decocting piece, add the ethanol of 8 times of amounts 60%, reflux 3 times, each 1.5 hours, merge alcohol reflux liquid, filter, concentrate standardize solution in the 100ml volumetric flask.

(4) 70% alcohol refluxs: take by weighing Flos Lonicerae, Radix Scrophulariae, Radix Achyranthis Bidentatae, each 10g of Herba Dendrobii pharmaceutical decocting piece, add the ethanol of 8 times of amounts 70%, reflux 3 times, each 1.5 hours, merge alcohol reflux liquid, filter, concentrate standardize solution in the 100ml volumetric flask.

2, the assay of chlorogenic acid, cinnamic acid in the Different Extraction Method sample liquid

The HPLC of chlorogenic acid measures

(1) chromatographic condition and system suitability test

The C18 post is a chromatographic column, methanol-0.1% formic acid solution gradient elution, and flow velocity: 1.0ml/min detects wavelength 327nm, and theoretical cam curve is not less than 1500.Gradient elution different time ratio sees Table 1-1.

Table 1-1 gradient elution table

(2) preparation of need testing solution

The accurate water of drawing is carried sample liquid 2ml, and adding distil water is diluted to 25ml, and is centrifugal, gets supernatant, uses for measuring.All the other 50%, 60%, 70% alcohol reflux liquid, the accurate respectively 2ml that draws adds 30% methanol and is diluted to 25ml, and is centrifugal, gets supernatant, uses for measuring.Measurement result sees Table 2.

The cinnamic acid assay

Chromatographic condition and system suitability test: the C18 post is a chromatographic column, and methanol-0.1% phosphoric acid solution (60: 40) is a mobile phase; 278nm detects wavelength, and theoretical cam curve is not less than 1500

The preparation of need testing solution: the accurate water of drawing is carried sample liquid 2ml, and adding distil water is diluted to 25ml, and is centrifugal, gets supernatant, uses for measuring.All the other 50%, 60%, 70% alcohol reflux liquid, the accurate respectively 10ml that draws adds 50% methanol and is diluted to 25ml, and is centrifugal, gets supernatant, uses for measuring.Measurement result sees Table 12.

Assay method: precision is measured above-mentioned reference substance and each need testing solution, injects chromatograph of liquid, measures, and calculates with one point external standard method, and measurement result sees Table 1-2.

The different extracting mode chlorogenic acids of table 1-2, cinnamic acid extracted amount be (n=5) relatively

3, the result of chlorogenic acid, cinnamic acid extraction yield compares (Q check) in the Different Extraction Method sample liquid

By experimental result as can be known: in alcohol extraction and traditional extraction process by water gained extracting solution, the extraction yield unknown significance difference of chlorogenic acid, but the extraction yield of cinnamic acid has significant difference concurrently, 60% alcohol reflux, 70% alcohol reflux, 50% ethanol and water extraction are relatively, see significant difference is arranged, determine to select 50%～70% ethanol for extracting solvent, adopt reflux extraction to extract.Assay sees Table 1-3,1-4.

Table 1-3 different solvents is to the extraction yield of chlorogenic acid

Multiple?Comparisons

Dependent?Variable：DATA

Table 1-4 different solvents is to the extraction yield of cinnamic acid

Multiple?Comparisons

Dependent?Variable：DATA

LSD

^*、The?mean?difference?is?significant?at?the?.05?level.

Comparative example 2

This example is the comparative study of water precipitating, heat treatment cold preservation technology and traditional alcohol precipitation process.

The present invention uses some polarity impurity component less than normal and be difficult to dissolving in aqueous solution, and with compositions such as the closely related composition cinnamic acid of drug effect, chlorogenic acids deliquescent preferably principle is arranged in alcoholic solution, reaches the purpose of crude separation by water precipitating.And traditional handicraft and patent publication No. CN1660314A are isolating at the beginning of realizing by precipitate with ethanol.So these two kinds of technologies just being separated the difference of front and back compares.

1, test method

Extracting honeysuckle, Radix Scrophulariae, Radix Achyranthis Bidentatae, each 20g of Herba Dendrobii merge, and add 8 times of amount 60% ethanol extractions 3 times, and each 1.5 hours, merging the ethanol extract decompression recycling ethanol and being concentrated into did not have the alcohol flavor, is settled to 250ml.The accurate 10ml that draws as the sample solution before separating, measures usefulness, two parts of remainder five equilibriums fully.Portion adds the dilution of isopyknic water for injection, more than the heating in water bath to 95 ℃, be incubated 30 minutes, cooling back cold preservation 12 hours, and taking-up, it is centrifugal that (5500rpm, 15min), supernatant is settled to 250ml.Another part adds 95% ethanol, makes to contain the alcohol amount and reach 80%, leaves standstill 12 hours, and centrifugal (5500rpm 15min), gets supernatant, reclaims ethanol, adds dehydrated alcohol and is settled to 250ml.5 parts of parallel tests.

2, the preparation of test liquid:

Sample before just separating: in the accurate 2ml to 25ml of the absorption measuring bottle, be settled to scale in the 10ml sample liquid that keeps sample certainly, shake up with distilled water diluting, standby.

Sample after the water precipitating: the accurate absorption in 4ml to the 25ml measuring bottle in the 250ml sample liquid, be settled to scale with distilled water diluting, shake up, standby.

Sample after the precipitate with ethanol: the accurate absorption in 4ml to the 25ml measuring bottle in the 250ml sample liquid, be settled to scale with the dehydrated alcohol dilution, shake up, standby.

3, detection method and result

Determination of chlorogenic acid: method is with " extracting the comparison of solvent and extracting method ".

The cinnamic acid assay: method the results are shown in Table 1-5 with " extracting the comparison of solvent and extracting method ".

The comparison (n=5) of chlorogenic acid, cinnamic acid content before and after table 1-5 just separates

4, water precipitating merges before and after the heat treatment cold preservation, and chlorogenic acid and cinnamic acid mean transferred rate are 92.2%, 85.8%.And after the precipitate with ethanol processing, chlorogenic acid and cinnamic acid mean transferred rate are 80.3%, 57.8%.The mean transferred rate was significantly greater than before and after therefore precipitate with ethanol is handled before and after water precipitating merged heat treatment cold preservation.

Comparative example 3

This example is regulated the extremely tart investigation of pH value relatively for extraction is preceding.

The present invention considers that organic acid substances such as the index composition cinnamic acid, chlorogenic acid of medicinal liquid are free state under acid condition, water solublity descends, and when adopting ethyl acetate extraction, transfers in the organic facies easily, therefore, the influence of solution pH value to index sexual element extraction yield investigated in test.

(1) test method

Extracting honeysuckle, Radix Scrophulariae, Radix Achyranthis Bidentatae, each 100g of Herba Dendrobii add 60% alcohol reflux 3 times, each 8 times of amounts, and each 1.5 hours, merge ethanol extract, behind the decompression recycling ethanol, water precipitating, heat treatment cold preservation, centrifugal, get supernatant 4000ml, standby.The respectively accurate supernatant 50ml that draws with the ethyl acetate extraction of 2 times of amounts 6 times, combining extraction liquid, behind the reclaim under reduced pressure ethyl acetate, waves to no ethyl acetate with jacket steam and to distinguish the flavor of.Behind the gained extract reclaim under reduced pressure ethyl acetate, being dissolved in water is settled to 25ml, standby.Parallel 5 parts.

(2) assay of index composition

Chlorogenic acid, cinnamic acid content assaying method are the same.The gained result is converted to the content of chlorogenic acid, cinnamic acid in the 25ml extract.List table 1-7 in.

The different pH value of table 1-7 are to the result (n=5) of chlorogenic acid, cinnamic acid content influence

Conclusion: when adopting ethyl acetate extraction among the present invention, remarkable to the influence of index sexual element extraction yield.Regulate pH value to acidity and can significantly improve active constituent content.

Comparative example 4

This example is an assay in the injecting Mailuoning lyophilizing:

Cinnamic acid is measured according to high performance liquid chromatography (appendix VI D).

Chromatographic condition and system suitability test octadecylsilane chemically bonded silica Wei ?fill agent; Methanol-0.1% phosphoric acid solution (54: 46) is a mobile phase; The detection wavelength is 278nm.Theoretical cam curve is calculated by the cinnamic acid peak should be not less than 1500.

It is an amount of that the preparation precision of reference substance solution takes by weighing the cinnamic acid reference substance, and solubilizer methanol-water (54: 46) is made the solution that every 1ml contains 260 μ g, promptly.

This product content under the content uniformity item is got in the preparation of need testing solution, mixing, get 0.2g, the accurate title, decide, and puts in the 50ml volumetric flask, solubilizer methanol-water (54: 46) dissolves and is diluted to scale, shake up, therefrom accurate again the absorption in 5ml to the 10ml volumetric flask, with above-mentioned solvent dilution to scale, shake up, promptly.

Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.

Chlorogenic acid is measured according to high performance liquid chromatography (appendix VI D).

Chromatographic condition and system suitability test octadecylsilane chemically bonded silica Wei ?fill agent, be mobile phase with acetonitrile-0.1% phosphoric acid solution (11: 89), the detection wavelength is 327nm.Number of theoretical plate is not less than 1500 by the chlorogenic acid peak.

It is an amount of that the chlorogenic acid reference substance is got in the preparation of reference substance solution, and accurate the title decides, and puts in the brown volumetric flask, and solubilizer acetonitrile-water (11: 89) is made the solution that contains 80 μ g among every 1ml.

This product content under the content uniformity item is got in the preparation of need testing solution, mixing, get 0.2g, the accurate title, decide, and puts in the brown measuring bottle of 50ml, solubilizer acetonitrile-water (11: 89) dissolves and is diluted to scale, shake up, therefrom accurate again the absorption in the brown measuring bottle of 1ml to 25ml, with above-mentioned solvent dilution to scale, shake up, promptly.

Three batches of MAILUONING lyophilizing finished products by the present invention's preparation (preparation example 1) the results are shown in the 1-6 table with commercially available MAILUONING assay.

Cinnamic acid, chlorogenic acid content compare (n=3) in table 1-6 freeze dried Mailuoning powder pin and the commercially available MAILUONING ZHUSHEYE

Conclusion: the present invention compares with commercially available MAILUONING, and the MAILUONING lyophilizing active constituent content of being produced obviously improves.

Experimental example 1

This example is a freeze dried Mailuoning powder for injection injection fingerprint pattern quality control method.

According to high performance liquid chromatography (an appendix VI of 2000 editions Chinese Pharmacopoeias D), and in conjunction with " Chinese medicine chromatographic fingerprinting experimentation technical manual (trying) " requirement.

Chromatographic condition and system suitability test

Instrument: Agilent 1100LC series, Agilent chemstation chromatographic work station

Chromatographic column: kromasil-5C ₁₈Post (250mm * 4.6mm, 5 μ m)

Mobile phase is the A:0.1% aqueous formic acid, B: acetonitrile, and flow velocity 1.0ml/min, according to the form below carries out gradient elution, detects wavelength 254nm, 35 ℃ of column temperatures.The gradient elution program is:

It is an amount of that the chlorogenic acid reference substance is got in the preparation of object of reference, with dissolve with methanol and dilution, makes every 1ml and contain the solution of 16.16 μ g as object of reference solution; It is an amount of to get the cinnamic acid reference substance, with dissolve with methanol and dilution, makes every 1ml and contains the solution of 14.44 μ g as object of reference solution.

One of this product is got in the preparation of test sample, adds the redistilled water dissolving, puts in the 10ml measuring bottle, and thin up is to scale, shake up, the accurate absorption gone up in liquid 0.2ml to the 25ml measuring bottle, adds redistilled water and is diluted to scale, filters with 0.45 μ m filter membrane, discard filtrate just, get subsequent filtrate, promptly.

The accurate need testing solution 20 μ l that draw of algoscopy inject chromatograph of liquid, and be 70min writing time, gradient equilibration time 10min.

[methodology checking]

Reliability for verification method, the regulation of " the Chinese medicine chromatographic fingerprinting experimentation technical manual (trying) " issued according to National Drug Administration is all investigated the precision of stability of sample, instrument, the repeatability of experimental technique etc.The result is all up to specification.

Stability test: get same need testing solution, detect at different time respectively, investigate the concordance of each chromatographic peak similarity, utilize Chinese Pharmacopoeia Commission " chromatographic fingerprints of Chinese materia medica similarity evaluation system " A version in 2004 to carry out similarity evaluation, each similarity numerical value sees Table 1.

Table 1 injecting Mailuoning stability test similarity data

Precision test: get same need testing solution, continuous sample introduction 5 times, investigate the concordance of each chromatographic peak similarity, utilize Chinese Pharmacopoeia Commission " chromatographic fingerprints of Chinese materia medica similarity evaluation system " A version in 2004 to carry out similarity evaluation, each similarity numerical value sees Table.

Table 2 injecting Mailuoning precision test similarity data

Replica test: get with 5 of a collection of finished products, make test sample by [preparation of test sample], detect with method, investigate the concordance of each chromatographic peak similarity, utilize Chinese Pharmacopoeia Commission " chromatographic fingerprints of Chinese materia medica similarity evaluation system " A version in 2004 to carry out similarity evaluation, each similarity numerical value sees Table.

Table 3 injecting Mailuoning repeatability similarity data

[blank assay]

Preparation method by preparation technology and need testing solution makes blank solution, and precision is drawn 20 μ l, injects chromatograph of liquid, measures according to condition under text [chromatographic condition] item, the results are shown in Figure 4.The result shows not interference measurement of blank.

[determining of writing time]

Press text [chromatographic condition], write down after 70 minutes, keep 50 minutes chromatograms (promptly writing down 120 minutes finger printing) of mobile phase constant rate record, see Fig. 5.The result shows, does not detect chromatographic peak in retention time 70-120 minute, so determine that be 70 minutes analysis time.

[finger printing and every technical parameter]

Collection of illustrative plates integral parameter: slope: 1; Peak width: 0.04; Smallest peaks area: 20; Minimum peak height: 1.5; Paddy paddy integration.

Software for calculation: adopt state-promulgated pharmacopoeia can write " the chromatographic fingerprints of Chinese materia medica similarity evaluation 2004A of system version ".

Finger printing: Fig. 1 is the typical finger printing of injecting Mailuoning, Fig. 2 is the gradient elution collection of illustrative plates of reference substance (chlorogenic acid), Fig. 3 is the gradient elution collection of illustrative plates of reference substance (cinnamic acid), Fig. 4 is the gradient elution collection of illustrative plates of blank solvent, and Fig. 5 is the finger printing of 2 hours injecting Mailuoning of record.

Parameter is provided with:

(1) with reference to spectrogram: it is with reference to spectrogram that this product adopts reference fingerprint (common pattern).

(2) time window width: 0.10

(3) data are sheared: blank assay is noiseless, so need not shear.

(4) correcting mode: the mode that adopts gradient elution, the chromatographic peak retention time may be inconsistent with corresponding chromatographic peak retention time in the reference fingerprint in the test sample finger printing, so similarity should not adopt automatic correcting mode when calculating, proofread and correct and should carry out multiple spot.

Proofread and correct determining of chromatographic peak: (the about 20.2min of retention time, 23.9min is 59.8min) for proofreading and correct chromatographic peak with 3 higher chromatographic peaks of Strong degree.So proofread and correct with these 3 chromatographic peaks when similarity is calculated.

(5) finger printing similarity result of calculation

The similarity result of calculation of 10 batches of injecting Mailuonings sees Table 4.

The similarity of table 410 batch injecting Mailuoning

Similarity meansigma methods: 0.987

Table 1-4 result show, the similarity meansigma methods of 10 batches of injecting Mailuonings is greater than 0.90, according to above result of the test, and according to the regulation of " Chinese medicine chromatographic fingerprinting experimentation technical manual (trying) ", the finger printing of tentative this product should be consistent with the injecting Mailuoning reference fingerprint, and the similarity value should be not less than 0.90.

Experimental example 2 pharmacodynamic studies

(1) injection MLN is to the influence of hemorheology of rat

1. method

60 of male SD rats, body weight 320g～350g is divided into 6 groups at random by body weight, 10 every group.(1) MLN small dose group: 16.5g/kg; (2) dosage group: 33g/kg among the MLN; (3) the heavy dose of group of MLN: 66g/kg; (4) MLN injection group: 3.3ml/kg; (5) Radix Salviae Miltiorrhizae Injection group: 2g/kg; (6) normal saline group: 5ml/kg.Each group is all by 5ml/kg intraperitoneal injection every day 1 time, successive administration 10 days.0.5h after the last administration, every Corium Mus is injection 0.1% epinephrine 0.1mg/kg down, totally 2 times, 2 minor tick 4h, and in the middle of 2 injections, place 4 ℃ of frozen water to soak 5min animal, pentobarbital sodium 30mg/kg ip anesthesia next day 1%, common carotid artery blood sampling 4ml, whole blood blood viscosity and packed cell volume are measured in 1% heparin-saline solution anticoagulant (180U).

2. result

The result shows: injection MLN 16.5g/kg, 33g/kg, three dosage groups of 66g/kg ip 10d can reduce the whole blood viscosity under rat 200/s, 30/s, 5/s, the 1/s shear rate, with the normal saline group significant difference (P＜0.05,0.01) are arranged relatively.Its effect is close with the action intensity of MLN injection and Radix Salviae Miltiorrhizae Injection.Show that MLN has the hemorheological effect of improvement, sees Table 3-1.

MLN is to the rheol influence of rat serum (X ± S) (n=10) for table 3-1 injection

^*P＜0.05, ^*Compare with the normal saline group P＜0.01

(2) injecting Mailuoning (below be abbreviated as MLN) is to the influence of rat platelet aggregation rate

1. method

48 of male SD rats, body weight 250g～280g is divided into 6 groups at random by body weight, 8 every group.(1) MLN small dose group: 16.5g/kg; (2) dosage group: 33g/kg among the MLN; (3) the heavy dose of group of MLN: 66g/kg; (4) MLN injection group: 3.3ml/kg; (5) Radix Salviae Miltiorrhizae Injection group: 2g/kg; (6) normal saline group: 5ml/kg.Each group is all by 5ml/kg intraperitoneal injection every day 1 time, successive administration 10 days.0.5h after the last administration is with 1% pentobarbital sodium 30mg/kg ip anesthesia, through common carotid artery blood sampling 3ml, 3.8% liquor sodii citratis anticoagulant.With blood and anticoagulant (9: 1) mixing, the centrifugal 10min of 1000rpm draws the milky platelet blood plasma (PRP) that is rich in upper strata; Remaining part is the centrifugal 10min of 3000rpm once more, and supernatant is and contains platelet blood plasma (PPP) less, transfers PRP with PPP, and making its platelet count is 2.0 * 10 ⁵Individual/mm ³According to the inductive maximum platelet aggregation rate of turbidimetry for Determination ADP.The final concentration of ADP in opacity tube is 0.146mg/ml.

2. result

The result shows: injection MLN 16.5g/kg, 33g/kg, three dosage groups of 66g/kg all can suppress the maximum platelet aggregation rate of rat, relatively have significant difference (P＜0.01) with the normal saline group.Its effect is close with the action intensity of MLN injection and Radix Salviae Miltiorrhizae Injection.Suppression ratio is respectively 39.3%, 51.7%, 64.9%, 45.2% and 56.2%.Show that injection MLN has the effect of anticoagulant, sees Table 3-2.

Table 3-2 injection MLN is to the influence of rat platelet aggregation (X ± S)

^*Compare with the normal saline group P＜0.01

(3) injection MLN is to the thrombotic influence of rat carotid artery-external jugular vein

1. method

60 of male SD rats, body weight 210g～220g is divided into 6 groups at random by body weight, 10 every group.(1) MLN small dose group: 16.5g (crude drug)/kg; (2) dosage group: 33g (crude drug)/kg among the MLN; (3) the heavy dose of group of MLN: 66g (crude drug)/kg; (4) MLN injection group: 3.3ml/kg; (5) Radix Salviae Miltiorrhizae Injection group: 2g/kg; (6) normal saline group: 5ml/kg.Each group is all by 5ml/kg intraperitoneal injection every day 1 time, successive administration 10 days.0.5h after the last administration with 1% pentobarbital sodium 30mg/kg ip anesthesia, separates right common carotid artery and left external jugular vein.In three sections polyethylene tubes, put into the 4 trumpeter's art silk threads that are about 5cm.(50U/ml) is full of the polyethylene tube chamber with heparin-saline.After an end of pipe inserts left external jugular vein, inject heparin 50U/kg from polyethylene tube, clamp tube wall.The other end of pipe is inserted right common carotid artery.Back opening blood flow is finished in operation, and then blood returns external jugular vein from the right common carotid artery polyethylene tube of flowing through.Open blood flow is middle Herba Clinopodii after 15 minutes.Take out silk thread rapidly and weigh, gross weight deducts silk thread and heavily is wet weight of thrombus.With 70 ℃ of oven dry of silk thread, taking-up is weighed, and gross weight deducts silk thread and heavily is the thrombosis dry weight.Calculate suppression ratio by following formula:

2. result

The result shows: injection MLN 16.5g/kg, 33g/kg, three dosage groups of 66g/kg all can reduce the rat carotid artery-wet weight of thrombus of external jugular vein blood flow bypass formation and the weight of dry weight, suppression ratio is respectively 12.7%, 26.0%, 38.6% (wet weight of thrombus), 20.8%, 39.6%, 47.9% (thrombosis dry weight) relatively has significant difference (P＜0.05,0.01) with the normal saline group.The action intensity of dosage group and MLN injection and Radix Salviae Miltiorrhizae Injection is close among the MLN.Show that injection MLN has antithrombotic effect, sees Table 3-3.

Table 3-3 injection MLN is to rat carotid artery-thrombotic influence of external jugular vein bypass (X ± S)

^*P＜0.05, ^*Compare with the normal saline group P＜0.01

(4) injection MLN is to the thrombotic influence of rabbit common carotid artery

1. method

36 of healthy white big ear rabbits, male and female half and half, body weight 1.9～2.2kg is divided into 6 groups at random by body weight, 6 every group.(1) MLN small dose group: 10g/kg; (2) dosage group: 20g/kg among the MLN; (3) the heavy dose of group of MLN: 40g/kg; (4) MLN injection group: 2ml/kg; (5) Radix Salviae Miltiorrhizae Injection group: 1.2g/kg; (6) normal saline group: 2ml/kg.Each group is all by 2ml/kg auricular vein every day administration 1 time, successive administration 3 days.0.5h after the last administration, 3% pentobarbital sodium 30mg/kg iv anesthesia separates bilateral common carotid arteries, 3cm closes right carotid with two bulldog clamp folders at interval, with No. 4 syringe needle extractions blood wherein, 0.9% sodium chloride injection flushing several is to the about 1ml of hydrogen peroxide of this section intra-arterial injection 10%, extract out behind the 15min, after the flushing for several times of 0.9% sodium chloride injection, extract syringe needle, the dry cotton ball hemostasis by compression, decontrol bulldog clamp, make blood flow logical again.The isometric bilateral common carotid arteries of cutting is weighed behind the 2h, measures its length, calculates wet weight of thrombus (mg), w/w index (mg/mg) by following formula.

Heavy (the mg)-left common carotid artery of wet weight of thrombus (mg)=right common carotid artery heavy (mg)

2. result

The result shows: injection MLN 10g/kg, 20g/kg, three dosage groups of 40g/kg all can reduce wet weight of thrombus and w/w index, suppression ratio is respectively 27.7%, 30.7%, 70.5% (wet weight of thrombus), 4.2%, 33.3%, 62.5% (w/w index) relatively has significant difference (P＜0.05,0.01) with the normal saline group.Show that injection MLN has antithrombotic effect, action intensity is suitable with the effect of MLN injection group.See Table 3-4.

Table 3-4 injection MLN is to the thrombotic influence of rabbit common carotid artery (X ± S)

^*P＜0.05, ^*Compare with the normal saline group P＜0.01

(5) injection MLN causes the influence of mouse death rate to arachidonic acid

1. method

180 of male KM mices, body weight 18g～22g is divided into 6 groups at random by body weight, 30 every group.(1) MLN small dose group: 33g/kg; (2) dosage group: 66g/kg among the MLN; (3) the heavy dose of group of MLN: 132g/kg; (4) MLN injection group: 6.6ml/kg; (5) Radix Salviae Miltiorrhizae Injection group: 4g/kg; (6) normal saline group: 10ml/kg.Each group is all by 10ml/kg intraperitoneal injection every day 1 time, successive administration 7 days.0.5h after the last administration, adopt reported method such as Kohler, give (iv) arachidonic acid (AA) 80mg/kg (the AA solution 0.16ml/10g of 5mg/ml) of mouse tail vein injection, breathing situation, the active state of observing mice immediately, and dead mouse number in the record 5min.The result causes that with iv AA the lung blood capillary forms thrombosis, the mortality rate that causes mouse breathing to die of exhaustion dying is represented statistical method x ²Check.

2. result

The result shows: two dosage groups of injection MLN 66g/kg, 132g/kg all can reduce arachidonic acid induced mice death toll, reduce mortality rate, relatively have significant difference (P＜0.05,0.01) with the normal saline group.Show that injection MLN has antithrombotic effect, suitable with the effect of MLN injection group.See Table 3-5.

Table 3-5 injection MLN causes the influence of mouse death rate to the intravenous injection arachidonic acid

^*P＜0.05 ^*Compare with the normal saline group P＜0.01

(6) injection MLN is to the influence of rat acute imperfection cerebral ischemia

1. method

56 of male SD rats, body weight 210g～220g is divided into 7 groups at random by body weight, 8 every group.(1) MLN small dose group: 16.5g/kg; (2) dosage group: 33g/kg among the MLN; (3) the heavy dose of group of MLN: 66g/kg; (4) MLN injection group: 3.3ml/kg; (5) Radix Salviae Miltiorrhizae Injection group: 2g/kg; (6) model group: NS 5ml/kg; (7) pseudo-operation group: NS 5ml/kg.Each group is all by 5ml/kg intraperitoneal injection every day 1 time, successive administration 10 days.0.5h after the last administration, anaesthetize with 1% pentobarbital sodium 30mg/kg ip, press the capable common carotid artery exclusion of literature method, (the only row operation of pseudo-operation group of ligation bilateral common carotid arteries, and not ligation tremulous pulse), broken end is got brain behind the 3h, claim that heavy, left and right half brain of full brain is heavy after, a left side half brain is put in 37 ℃ of calorstats and is dried to constant weight, calculates cerebral index, brain water content by following formula.

Right half brain-30 ℃ freezing 2h, the parallel coronalplane of scalpel is cut into 5～6 of the thick sections of 1～2mm, in the 2% red tetrazolium buffer (pH7.4) in 37 ℃ of waters bath with thermostatic control dyeing 15min after, take by weighing red dying (not infarct) part weight respectively and do not dye (infarct) part weight, be calculated as follows the infarct size percentage rate.

2. result

The result shows: injection MLN 16.5g/kg, 33g/kg, three dosage groups of 66g/kg all can reduce infarct size, cerebral index, the brain water content of rat acute imperfection cerebral ischemia, relatively have significant difference (P＜0.05,0.01) with model group.Its effect is close with MLN injection 3.3ml/kg, Radix Salviae Miltiorrhizae Injection 2g/kg.Show that injection MLN has anti-acute cerebral ischemia effect, sees Table 3-6.

Table 3-6 injection MLN is to the influence of rat acute imperfection cerebral ischemia (X ± S)

^ΔP＜0.05, ^{The Δ Δ}Compare with pseudo-operation group P＜0.01

^*P＜0.05, ^*Compare with model group P＜0.01

(7) injection MLN is to the influence of chmice acute cerebral ischemia mortality rate

1. method

70 of male KM mices, body weight 19g～22g is divided into 7 groups at random by body weight, 10 every group.(1) MLN small dose group: 33g/kg; (2) dosage group: 66g/kg among the MLN; (3) the heavy dose of group of MLN: 132g/kg; (4) MLN injection group: 6.6ml/kg; (5) Radix Salviae Miltiorrhizae Injection group: 4g/kg; (6) model group: 10ml/kg; (7) sham operated rats.Each group is all by 10ml/kg intraperitoneal injection every day 1 time, successive administration 7 days.0.5h after the last administration, the shallow fiber crops of ether, 4-0 silk thread ligation bilateral common carotid arteries, time-to-live in the record mice 5min (surpassing 5min, meter 300s) (the per minute breathing is less than or equals 5 times for dead).The sham operated rats operation technique is identical, but not ligation common carotid artery.

2. result

The result shows: injection MLN 33g/kg, 66g/kg, 132g/kg, MLN injection 6.6ml/kg, Radix Salviae Miltiorrhizae Injection 4g/kg can resist because of the chmice acute cerebral ischemia due to the ligation bilateral common carotid arteries, obviously prolong the mice time-to-live (P＜0.05,0.01).Show that injection MLN has treating cerebral ischemia, sees Table 3-7.

Table 3-7 injection MLN is to the influence of chmice acute cerebral ischemia time-to-live (X ± S)

^{The Δ Δ}Compare with pseudo-operation group P＜0.01

^*P＜0.05, ^*Compare with model group P＜0.01

(8) injection MLN is to the effect of isolated rat vascular smooth muscle

1. method

1.1 sample disposal

Select cleaning level SD rat for use, body weight 250～300g, male and female are regardless of.Adopt the Mus decapitator with the rat sacrificed by decapitation, open the thoracic cavity rapidly, get its thoracic aorta, place to pass to (95% O ₂+ 5%CO ₂, flow 2L/min) and (K-H physiological liquid composition [g/L]: NaCl6.92, KCl 0.35, CaCl for the saturated cold Krebs-Henseleit physiological liquid of mist ₂0.28, KH ₂PO ₄0.16, MgSO ₄7H ₂O 0.46, NaHCO ₃2.1 Glucose 2.0) in, wipe out tunica adventitia of artery fat and connective tissue gently, and be cut into the wide vascular ring number of samples of 3mm section.Vascular ring is run through vessel lumen with two miniature hooks of rustless steel, and specimen hangs in the bath pipe of containing 15ml KH liquid, and two ends are connected in tonotransducer respectively and bathe on the fixation hook of pipe bottom, continue to feed mist (95%O ₂+ 5%CO ₂, flow 2L/min), keep temperature (37 ± 0.5) ℃.Link to each other the variation of record antiotasis with computer through Medlab-U/8c bio signal acquisition processing system (analog-digital converter).Each vascular ring does not add the tension force temperature and bathes 30min after being suspended on and bathing in the pipe, gives the preload of 2.0g then, and temperature is bathed 1h.Constantly adjust tension force during temperature is bathed, make it to keep stable, every 15min changes a K-H liquid.1.2 injection MLN is to the influence of arterial ring contractile response due to the KCl

(20,40, about 15min (being as the criterion to reach maximum collapse at every turn) is observed in 60mmol/L) stimulation at every turn to add KCl liquid with the accumulative total concentration method.Get the amount effect curve that KCl shrinks aortic annulus.After each section vasoconstriction reaches stable maximum collapse platform, wash arterial ring (every 15min changes 37 ℃ of K-H liquid, 15ml/ time) repeatedly with K-H liquid, treat that its tension force recovers normal back 60min, totally adding KCl liquid again stimulates.When each blood vessel stimulates when stablize the caused shrinkage amplitude difference of promptly continuous 2 same stimulations＜10% o'clock, the effect of observation pharmaceutical intervention to KCl.

After each arteries is stablized 60min with the flushing of K-H liquid, be divided into MAILUONING ZHUSHEYE I, MAILUONING ZHUSHEYE II, MLN I, MLN II, MLN III, MLN IV group, add medicine (adopting K-H liquid to prepare) each 1ml of solution that has been formulated as variable concentrations respectively, make to bathe to be following final concentration in the pipe: 1. MAILUONING ZHUSHEYE I organizes: 0.0125ml stock solution/ml; 2. MAILUONING ZHUSHEYE II organizes: 0.025ml stock solution/ml; 3. MLN I organizes: 0.03125g (crude drug)/ml; 4. MLN II organizes: 0.0625g (crude drug)/ml; 5. MLN III organizes: 0.125g (crude drug)/ml; 6. MLN IV organizes: 0.25g (crude drug)/ml.Behind the 20min, accumulative total adds KCl liquid, and (20,40,60mmol/L) stimulation is observed variable concentrations MLN to the pressor influence of KCl after the administration.

After having observed the intervention effect of medicine, wash arterial ring repeatedly, treat that its tension force recovers normally back 60min with K-H liquid, and reuse accumulative total concentration method adding KCl liquid (20,40,60mmol/L) stimulate, observation tremulous pulse this moment is to the reactivity of KCl.

1.3 injection MLN is to the influence of arterial ring contractile response due to the norepinephrine

Add norepinephrine NA (10 with the accumulative total concentration method ^-7～10 ^-5Mmol/L) stimulate each about 15min (being as the criterion) that observes to reach maximum collapse at every turn.Get the amount effect curve that NA shrinks aortic annulus.After vasoconstriction reaches stable maximum collapse platform, wash arterial ring (every 15min changes 37 ℃ of K-H liquid, 15ml/ time) repeatedly with K-H liquid, treat that its tension force recovers normal back 60min, totally adds norepinephrine NA (10 again ^-7～10 ^-5Mmol/L) stimulate.When blood vessel stimulates when stablize the caused shrinkage amplitude difference of promptly continuous 2 same stimulations＜10% o'clock, the effect of observation pharmaceutical intervention to NA.

After tremulous pulse is stablized 60min with the flushing of K-H liquid, be divided into MAILUONING ZHUSHEYE I, MAILUONING ZHUSHEYE II, MLN I, MLN II, MLN III, MLN IV group, add medicine (adopting K-H liquid to prepare) each 1ml of solution that has been formulated as variable concentrations respectively, make to bathe to be following final concentration in the pipe: 1. MAILUONING ZHUSHEYE I organizes: 0.0125ml stock solution/ml; 2. MAILUONING ZHUSHEYE II organizes: 0.025ml stock solution/ml; 3. MLN I organizes: 0.03125g (crude drug)/ml; 4. MLN II organizes: 0.0625g (crude drug)/ml; 5. MLN III organizes: 0.125g (crude drug)/ml; 6. MLN IV organizes: 0.25g (crude drug)/ml.Behind the 20min, accumulative total adds norepinephrine NA (10 after the administration ^-7～10 ^-5Mmol/L) stimulate, observe variable concentrations MLN the pressor influence of NA.

After having observed the intervention effect of medicine, wash arterial ring repeatedly with K-H liquid, treat that its tension force recovers normal back 60min, reuse accumulative total concentration method adds norepinephrine NA (10 ^-7～10 ^-5Mmol/L) stimulate, observation tremulous pulse this moment is to the reactivity of NA.

2. result

2.1 injection MLN is to the influence of arterial ring contractile response due to the KCl

The result shows, MAILUONING ZHUSHEYE 0.0125ml stock solution/ml～0.025ml stock solution/ml and injection MLN0.03125g (crude drug)/ml～0.25g (crude drug)/ml concentration dependent ground suppresses KCl (20～60mmol/L) contractions to arterial ring (p＜0.05,0.01).MAILUONING ZHUSHEYE makes the caused arterial ring contraction of 60mmol/LKCl amplitude descend 11.11%, 16.67% respectively.Injection MLN makes the caused arterial ring contraction of 60mmol/LKCl amplitude descend 12.59%, 13.60%, 50.00%, 74.38% respectively.The results are shown in Table 3-8.

Table 3-8 injection MLN is to the influence of arterial ring contractile response due to the KCl (X ± S)

With comparison before the administration, ^*P＜0.05, ^*P＜0.01; Compare with MLN I, ^{The Δ Δ}P＜0.01; Compare with MLN II,

P＜0.05, P＜0.01; Compare with MLN III, ^{◇ ◇}P＜0.01;

2.2 injection MLN is to the influence of arterial ring contractile response due to the norepinephrine

The result shows that MAILUONING ZHUSHEYE 0.0125ml stock solution/ml～0.025ml stock solution/ml and injection MLN0.03125g (crude drug)/ml～0.25g (crude drug)/ml concentration dependent ground suppresses NA (10 ^-7～10 ^-5Mmol/L) to the contraction (p＜0.05,0.01) of arterial ring.MAILUONING ZHUSHEYE makes 10 ^-5The caused arterial ring contraction of mmol/L NA amplitude descends 15.48%, 28.57% respectively.Injection MLN makes 10 ^-5The caused arterial ring contraction of mmol/LNA amplitude descends 10.18%, 23.35%, 56.41%, 78.65% respectively.The results are shown in Table 3-9.

Table 3-9 injection MLN is to the influence of arterial ring contractile response due to the norepinephrine (X ± S)

P＜0.05,

P＜0.01; Compare with MLN III, ^{◇ ◇}P＜0.01;

Experiment conclusion:

Injection MLN has blood viscosity lowering, improves hemorheology, anticoagulant, antithrombotic form and vasodilative effect, and cerebral ischemia is had the certain protection effect.Injection MLN pharmacological action is remarkable, obviously is better than commercially available MLN group in the part experiment.

The test of experimental example 3 general pharmacologies

1. intravenous injection injection MLN 33,132,264g/kg (crude drug amount) all do not cause mice sialorrhea, amyostasia phenomenon; Mice general behavior such as posture, gait and autonomic activities are not had obvious influence, compare there was no significant difference (P＞0.05) with the blank group;

2. intravenous injection injection MLN 33,132,264g/kg (crude drug amount) and immediately behind the lumbar injection pentobarbital sodium, its sleeping rate, dropping asleep latency and the length of one's sleep and blank group comparison there was no significant difference (P＞0.05);

3. behind intravenous injection injection MLN 33,132, the 264g/kg (crude drug amount), each the outstanding tail time of stopping and blank group of organizing mice compares no significant difference (p＞0.05);

4. behind intravenous injection injection MLN 33,132, the 264g/kg (crude drug amount), each is organized landing number of times and the blank group of mice in the gimbal lever and compares no significant difference (p＞0.05);

5. behind intravenous injection injection MLN 33,132, the 264g/kg (crude drug amount), each organizes mice landing number of elements and blank group no significant difference (p＞0.05) relatively from the hang plate.

6. behind intravenous drip injection MLN 3.3,13.2, the 26.4g/kg (crude drug amount), frequently to domesticated dog blood pressure, electrocardiogram, breathing

Rate and amplitude of respiration and blank group be no significant difference (p＞0.05) relatively.

In sum, injection MLN above-mentioned be subjected in the amount of reagent scope central nervous system of mice, domesticated dog cardiovascular system and respiratory system do not had obviously substantially influence.

Experimental example 4 acute toxicity tests

The maximum dosage-feeding test of intravenously administrable and intraperitoneal administration has been carried out in experiment to mice and rat.The result there is no mice and rat after administration abnormal conditions take place.If the clinical adult one consumption per day 200g/60kg of injection MLN (pressing the crude drug amount calculates), then

1. the mice maximum dosage-feeding of twice intravenously administrable is 2000g/kg in one day, is 606 times of clinical adult's one consumption per day;

2. the mice maximum dosage-feeding of twice intraperitoneal injection is 2000g/kg in one day, is 606 times of clinical adult's one consumption per day;

3. the rat maximum dosage-feeding of an intravenously administrable is 320g/kg, is 97 times of clinical adult's one consumption per day;

4. the rat maximum dosage-feeding of twice intraperitoneal injection is 960g/kg in one day, is 290 times of clinical adult's one consumption per day.

Show that injection MLN intravenously administrable degree of safety is bigger.

Experimental example 5 long term toxicity tests

(1) the long poison of rat

Give rats by intraperitoneal injection 12 week and 2 weeks of recovery continuously with injection MLN 33g/kg, three dosage groups of 100g/kg, 200g/kg (pressing the crude drug amount calculates).The result:

1. the rat general signs is good, and body weight, food ration are normal, the relatively more basic there was no significant difference of hematology, blood parameters and organ coefficient and matched group;

2. histological examination result shows: injection MLN high dose group is to heart, liver, spleen, lungs, kidney, stomach, duodenum, ileum, colon, pancreas, thyroid, thymus, adrenal gland, brain, spinal cord, optic nerve, hypophysis cerebri, lymph node, bladder, prostate, testis, epididymis, ovary, uterus, breastbone and bone marrow etc.

The property 23 internal organs, 22 internal organs of ♀ the pathology performance similar to matched group, do not look into after the drug withdrawal yet and see emerging tardy sexually transmitted disease (STD) change.

In sum, injection MLN does not have the toxic action of the property accumulated for a long time substantially to rat.

(2) the long poison of dog

Give Beagle dog forelimb subcutaneous 12 week and 2 weeks of recovery of cephalic vein administration continuously with injection MLN 13.2g (crude drug amount)/kg, 66g (crude drug amount)/kg and 132g (crude drug amount)/three dosage groups of kg, dog general signs, body weight, food-intake, hematological indices, routine urinalysis, blood parameters and organ coefficient do not have obvious influence substantially as a result; Histological examination shows that also high dose group is to the dog heart, liver, spleen, lungs, kidney, esophagus, stomach, duodenum, jejunum, ileum, colon, pancreas, salivary gland, gallbladder, trachea, thyroid, parathyroid gland, thymus, the adrenal gland, brain, cerebellum, brain stem, the cervical part of esophagus spinal cord, chest section spinal cord, waist section spinal cord, optic nerve, hypophysis cerebri, sciatic nerve, lymph node, bladder, prostate, testis, epididymis, ovary, the uterus, breastbone and bone marrow, mammary gland, administration part (vein) etc.

The property, each 35 internal organs of ♀ or organize and do not see tangible histology's pathological change.Show that injection MLN is less to dog toxicity through intravenously administrable.

Experimental example 6 safety testings

1. the hemolytic result of the test shows, injection MLN (lot number: 040608) do not see haemolysis and agglutination;

2. Cavia porcellus systemic anaphylaxis result of the test shows, injection MLN (lot number: 040608) anaphylaxis pass the test;

3. rabbit blood vessel irritation result of the test shows, injection MLN (lot number: 040608), behind intravenously administrable, do not look into and see that any blood vessel irritation changes, prompting injection MLN (lot number: 040608) to the basic nonirritant reaction of blood vessel by the injection site;

4. rabbit muscle irritation result of the test shows, injection MLN (lot number: 040608), behind the quadriceps femoris drug administration by injection, the injection site is not looked into and is seen that any zest changes, prompting injection MLN (lot number: 040608) to the basic nonirritant effect of rabbit quadriceps femoris;

5. rat passive cutaneous anaphylaxis test of the same race (PCA) result shows, injection MLN (lot number: 040608) should belong to the medicine that can not bring out cutaneous passive anaphylaxis, advise clinical continuation observation.

In sum, injection MLN meets the security requirement of injection, but injection for intravenous uses.

Annotate: MLN is the abbreviation of MAILUONING, and this experiment MAILUONING lyophilizing use amount is all in the crude drug amount, and used lyophilizing is preparation example 1 pilot product.

Claims

1, a kind of preparation method of injecting Mailuoning is characterized in that it being to make by following steps:

(7) filtrate is propped up packing with 5～8ml/, half moulding plug, and the sabot inlet carries out lyophilizing by freeze-drying curve, and vacuum is put in tamponade after the lyophilizing, and outlet rolls aluminium lid, and check is labelled, packing, promptly.

2, method according to claim 1 is characterized in that the drying means in the described step (5) is a spray drying or vacuum drying a kind of.

3, method according to claim 2 is characterized in that it being to make by following steps:

4, method according to claim 3 is characterized in that: the adding excipient is a mannitol, and consumption is that 0.3g/ props up.