CN100522139C - 'Mailuoning' injection and preparation method - Google Patents
'Mailuoning' injection and preparation method Download PDFInfo
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- CN100522139C CN100522139C CNB2004101029484A CN200410102948A CN100522139C CN 100522139 C CN100522139 C CN 100522139C CN B2004101029484 A CNB2004101029484 A CN B2004101029484A CN 200410102948 A CN200410102948 A CN 200410102948A CN 100522139 C CN100522139 C CN 100522139C
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Abstract
The present invention involving a kind of chinese medicine 'mailuoning' injection and its preparation method, which belongs to chinese medicine preparation fields. Firstly, obtaining the medicine effective composition of the medicine by the process including primary alcohol extrating which uses 0.5%-99.5% ethanol aqueous solution,gelatiin precipitation which uses 0.5%-10% gelatin aqueous solution and secondary alcohol sinking which should make the alcohol of supernatants is 75-85% from achyranthes bidentata,radix scrophulariae,dendrobium and honeysuckle orderly,then through the process of acetic acid and ecetic ester extraction and separation,the 'mailuning' injection will be made after the last process of freezing and drying.This invention product improves the purity greatly and removes the tannin,polysaccharide,protein and phlegmatic etc impurity,compare with the traditional liquid injection,powder injection prolongs the validity of the medicine and makes it have better stability,method and technology is simple,which improves the yield and validity.
Description
Technical field:
The present invention relates to a kind of Chinese medicine injection " MAILUONING " injection and preparation method thereof, belong to field of traditional Chinese.
Background technology:
" MAILUONING " is the theory according to traditional Chinese medical science clearing away heat and nourishing YIN, activating blood circulation to dissipate blood stasis, adopt rare Chinese medicine through extracting the refining compound Chinese medicinal preparation that forms, be used for the treatment of angiopathy on every side such as cerebral infarction sequela, cardiovascular disease, phlebothrombosis, large artery trunks thrombosis, vasculitis, in Chinese patent medicine, have consequence.
Four flavor Chinese medicines are Radix Achyranthis Bidentatae, Radix Scrophulariae, Herba Dendrobii and Flos Lonicerae in the MAILUONING prescription, and wherein Radix Achyranthis Bidentatae main chemical triterpenoid saponin generates oleanolic acid after the hydrolysis, and contains ecdysterone and inokosterone.In addition, still contain oligosaccharide, polysaccharide etc.Wherein one of main component is an ecdysterone, and ecdysterone is soluble in methanol, ethanol, ethyl acetate.Pharmacological evaluation shows the effect that animal is had blood sugar lowering and cholesterol.
The Radix Scrophulariae main chemical is four types of the plain glycosides of iridoid, phenylpropyl alcohol, triterpene saponin and organic aromatic acids.Wherein mainly containing effective constituent is cinnamic acid, is dissolved in alcohol, is soluble in ethyl acetate.Find to have function of increasing leukocyte during cinnamic acid is clinical, zoopery in recent years confirms to truly have leukogenic effect, zoopery also to find to improve peripheral leukocytes and platelet count etc.
Herba Dendrobii is the processed goods of Herba Dendrobii, sieve river Herba Dendrobii, dendrobium wilsonii Rolfe, thin footpath Herba Dendrobii etc.Mainly contain dendrobine, Herba Dendrobii time alkali, 6-hydroxydendrobine.It is reported, still separate five kinds of quartermary ammonium alkaloidses.Also separate aiapin, escoparone etc.Alkaloid is soluble in ethanol, methanol; Escoparone is soluble in methanol, ethanol, chloroform etc.Wherein dendrobine has analgesic effect, and blood pressure and breathing are had inhibitory action etc.; Escoparone has hypotensive activity.0.4-10mg/kg quiet notes or duodenal administration all have remarkable antihypertensive effect to general anesthesia or local anaesthesia rat, cat and rabbit.This effect by six hydrocarbon quaternary amines and atropine blocking-up, is not strengthened by phentolamine, can not be to adrenolytic boosting.Do vertebral artery when injection with the quiet injection volume of 1/50-1/10, the quiet notes of blood pressure lowering intensity and full dose point out that its hypotensive effect may be for central about equally.Escoparone has potentiation to the contractility of the Cor Leporis on the throne and the cat heart.Escoparone also can make the extrasomatic rabbit heart coronary flow increase.
Mainly contain in the Flos Lonicerae luteolin, chlorogenic acid, isochlorogenic acid, luteolin-7-glycoside (galuteolin), 5-hydroxyl-3 ', 4 ', 7-trimethoxy flavone etc.Above composition chlorogenic acid, dissolubility is bigger in hot water, is soluble in ethanol, and the atomic ethyl acetate that is dissolved in has extensively antibacterial action, hemostasis is arranged, increase leukocyte and antivirus action; Luteolin is dissolved in ethanol and alkaline solution; Galuteolin is slightly soluble in water, methanol, ethanol, dissolves in hot water, hot methanol and ethanol.To cardiovascular effect: luteolin, this rhinoceros grass glycoside have the rabbit of reduction experiment type animal atherosis in cholesteric effect, subcutaneous injection has the effect that strengthens blood capillary, wherein luteolin can make arterial pressure increase and reduce venous pressure, coronary blood flow increasing.
Traditional " MAILUONING " injection preparation technology complexity, the method effective component extracting of employing decocting in water precipitate with ethanol uses ethyl acetate extraction to purify then; For example, Chinese patent CN92107848.X discloses a kind of " process of MAILUONING intravenous fluid ", this method according to certain weight ratio compatibility, is positioned over Flos Lonicerae, Radix Achyranthis Bidentatae, Radix Scrophulariae, Herba Dendrobii and adds water logging in the reactor and boil collecting decoction behind twice decocting after fully mixing, use ethanol precipitation, finally contain the alcohol amount and be 70-75%, pure liquid is concentrated, remove surplus alcohol, with ethyl acetate extraction 7-10 time, obtain the effective ingredient of medicine.The MAILUONING ZHUSHEYE of this method preparation is used for thromboangiitis obliterans 157 examples, cure rate 48.4%, obvious effective rate 45.85%, total effective rate 97.5%; Be used for cerebral thrombosis and sequela 52 examples, basic cure rate 20.5%, obvious effective rate 38.5%, improvement rate 30.8%, total effective rate 84.74%.
Desirable medicine as thrombotic disease, the MAILUONING product that above-mentioned preparation method obtains still exists Impurity removals such as tannin not thorough, with ethyl acetate extraction, still have tannin can be extracted in the ethyl acetate, the unbalanced problem of active constituent content influences the oeverall quality of medicine, if further improvement is done in the extraction of effective ingredient, improve the purity of medicine, effective ingredient is done more specifically control, then can significantly improve drug quality, strengthen drug effect.Await doing further improvement or develop new process.
Meanwhile, traditional MAILUONING is an intravenous fluid, and inevitably there is poor stability in itself, meets light and is prone to effective ingredient decline, is difficult for the drawback of long term storage, if be prepared as injectable powder, then can help the problems referred to above to solve.Injectable powder is the abbreviation of injectable sterile powder, all to thermally labile or in aqueous solution labile medicine, as some antibiotic, enzyme preparation and biochemical product, owing to can not make general water soluble parenteral solution or be not suitable for heat sterilization, long term storage, all need make injectable sterile powder, i.e. injectable powder.Because it is not satisfactory that the stability of injection is compared than other dosage forms, in recent years, for improving the stability of Chinese medicine, existing part Chinese medicine injection is developed into injectable powder and uses.
The production of injectable powder must be carried out in sterilizing room, presses the different in kind of medicine, crude drug can be refined into sterilized powder, and direct packaging seals in cleaning sterilization bottle or ampoule and makes under aseptic condition; Or just medicine is made aseptic aqueous solution, with sterile working's fill, after lyophilization, forms in sealed under aseptic conditions.Traditionally, the former claims sterilized powder direct packaging method, and manufactured goods are aseptic subpackaged product; The latter claims the aseptic aqueous solution freeze-drying, and manufactured goods are freeze-dried product.At present, the Chinese medicine powder injection is many with freeze-dried products.
The injectable powder prescription is consistent with aqueous solution for injection, should meet " in the Chinese pharmacopoeia about every regulation of injectable drug and every check criteria of injectable sterile powder.
Freeze-drying is that medicinal liquid earlier is frozen into solid after aseptic subpackaged, then under the condition of low temperature and fine vacuum, makes the water sublimed in the medicinal liquid and removes, and under aseptic condition, adds a cover rubber closure, the aluminium lid sealing is made.Use this method and can avoid medicine rotten because of hyperpyrexia decomposes, the resulting product quality is relaxed and comfortable, adds dissolving rapidly behind the water, and water content is low, generally in the 1-3% scope, helps medicine and stores.
Summary of the invention:
The object of the present invention is to provide a kind of Chinese medicine " MAILUONING " injection.
Another object of the present invention is to provide the preparation method of a kind of Chinese medicine " MAILUONING " injection.
Traditional Chinese medicine preparation " MAILUONING " injection is a Chinese medicine compound, by Radix Achyranthis Bidentatae, Radix Scrophulariae, Herba Dendrobii and Flos Lonicerae 4 flavor Chinese medicine assembly; Extract its effective ingredient by the decocting in water precipitate with ethanol, after it is ethyl acetate extraction obtains purified product, formulated.In the extraction of its effective ingredient, impurity such as removal tannin are not thorough, influence the quality of injection.
'Mailuoning ' injection of the present invention, contain the raw material components of following weight portion:
Radix Achyranthis Bidentatae 100-1000 Radix Scrophulariae 100-1000
Herba Dendrobii 100-1000 Flos Lonicerae 100-1000
Above-mentioned Radix Achyranthis Bidentatae, Radix Scrophulariae, Herba Dendrobii and Flos Lonicerae obtain effective ingredient through the two-stage alcohol extraction, separate through ethyl acetate extraction again, obtain effective ingredient.
Wherein, the alcoholic solution that the one-level precipitate with ethanol uses is the ethanol water that contains alcohol amount 0%-99.5%, and the alcohol amount that contains of the supernatant during the secondary precipitate with ethanol is 75-85%.
Above-mentioned ethyl acetate extraction separating step also can the macroporous resin purification step replace, and realizes the object of the invention equally.
The invention described above method also can increase by a gelatin settling step between one-level alcohol extraction and a precipitate with ethanol, help the extraction of effective ingredient more, in the case, still can realize purpose of the present invention.
The inventive method can also be that above-mentioned Radix Achyranthis Bidentatae, Radix Scrophulariae, Herba Dendrobii and Flos Lonicerae are obtained effective ingredient through alcohol extraction, handles through aqueous gelatin solution or ZTC clarifier again, uses macroporous resin to purify at last, obtains effective ingredient.
The inventive method further can be above-mentioned Radix Achyranthis Bidentatae, Radix Scrophulariae, Herba Dendrobii and Flos Lonicerae to be carried through water obtain effective ingredient, handles through the ZTC clarifier again, uses macroporous resin to purify behind the supernatant precipitate with ethanol, obtains effective ingredient.
In the above-mentioned 'Mailuoning ' injection, escoparone content is measured according to high performance liquid chromatography (appendix VID of Chinese Pharmacopoeia version in 2000).
Chromatographic condition and system suitability test: with octadecylsilane chemically bonded silica is filler; Acetonitrile-water (1:4) is mobile phase; Detect wavelength 343nm, number of theoretical plate calculates by the escoparone peak should be not less than 2000.
The preparation of reference substance solution: it is an amount of that precision takes by weighing the escoparone reference substance, adds methanol and make the solution that contains 5 μ g among every 1ml, promptly.
The preparation of need testing solution: get product 50ml of the present invention, add sodium chloride 5g, use chloroform extraction 3 times, each 30ml, combined chloroform liquid, put evaporate to dryness in the water-bath, residue adds 60% methanol makes dissolving, and is transferred in the 25ml measuring bottle, add 60% methanol and be diluted to scale, shake up, filter with 0.45 μ m microporous filter membrane, promptly.
Algoscopy: precision is measured reference substance solution and each 20 μ l of need testing solution respectively, injects chromatograph of liquid, measures, promptly.
The every 1ml of product of the present invention contains Herba Dendrobii with escoparone (C
11H
10O
4) meter, escoparone content is 2.0-5.5 μ g/mL.
In the above-mentioned 'Mailuoning ' injection, determination of total flavonoids is as follows:
The preparation of reference substance solution: precision takes by weighing 120 ℃ of control substance of Rutin 10mg that are dried to constant weight, puts in the 100ml measuring bottle, and it is an amount of to add 50% methanol, and jolting makes dissolving, and is diluted to scale, shakes up, and promptly gets (containing rutin 0.1mg among every 1ml).
The preparation of standard curve: precision is measured reference substance solution 1.0,2.0,3.0,4.0,5.0ml, puts respectively in the 10ml measuring bottle, respectively adds 50% methanol to 5ml, add 5% sodium nitrite solution 0.3ml, shake up, placed 6 minutes, add 10% aluminum nitrate solution 0.6ml, shake up, placed 6 minutes, and added 8.6% sodium hydroxide solution 3ml, add 50% methanol again and be diluted to scale, shaking up, is blank with the reagent corresponding.According to spectrophotography (appendix VB of Chinese Pharmacopoeia version in 2000), measure trap at the wavelength place of 510nm, be vertical coordinate with the trap, concentration is abscissa, the drawing standard curve.
Algoscopy: precision is measured this product 2ml, puts in the 25ml measuring bottle, adds 50% methanol and is diluted to scale, shakes up.Precision is measured 3ml, puts in the 10ml measuring bottle, adds 50% methanol and is diluted to scale, shakes up, as blank; Precision is measured 3ml in addition, puts in the 10ml measuring bottle, and the method under the sighting target directrix curve preparation from " adding 50% methanol to 5ml ", is measured trap in accordance with the law immediately, reads the weight that is equivalent to rutin the need testing solution from standard curve, calculates, promptly.
The every 1ml of product of the present invention contains total flavones with rutin (C
27H
30O
16) meter, general flavone content is 2.0-3.5mg/mL.
'Mailuoning ' injection of the present invention is measured according to high performance liquid chromatography (2000 editions appendix VID of Chinese Pharmacopoeia), and mobile phase is methanol-water-glacial acetic acid (45:55:0.2), detects wavelength 243nm, and number of theoretical plate is pressed the cinnamic acid peak and calculated 〉=1000; Mobile phase is methanol-water-glacial acetic acid (15:85:1), detects wavelength 330nm, and number of theoretical plate is pressed the chlorogenic acid peak and calculated 〉=1000.Specific as follows:
(1) get product 10ml of the present invention, add ammonia solution and regulate pH value, use chloroform extraction 2 times to neutral, each 15ml, combined chloroform liquid is put evaporate to dryness in the water-bath, and residue adds methanol 1ml makes dissolving, as need testing solution.Other gets the escoparone reference substance, adds methanol and makes the solution that contains 0.1mg among every 1m, in contrast product solution.According to thin layer chromatography (" appendix VIB of Chinese pharmacopoeia version in 2000) test, measure each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, with cyclohexane extraction-chloroform-ethyl acetate (10:15:15) is developing solvent, launch, take out, dry, put under the ultra-violet lamp (365nm) and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the fluorescence speckle of same color.
(2) sample thief is as need testing solution.It is an amount of that other gets the cinnamic acid reference substance, adds methanol and make the solution that contains 50 μ l among every 1ml, in contrast product solution.Measure according to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2000 D), mobile phase is methanol-water-glacial acetic acid (45:55:0.2), detects wavelength 243nm, and number of theoretical plate calculates by the cinnamic acid peak should be not less than 1000.Have in the test sample chromatogram and the corresponding to chromatographic peak of the retention time of reference substance.
(3) it is an amount of to get the chlorogenic acid reference substance, adds methanol and makes the solution that contains 50 μ g among every 1ml, in contrast product solution.According to high performance liquid chromatography (" an appendix VI of Chinese pharmacopoeia version in 2000 D), the need testing solution of getting under reference substance solution and discriminating (2) item is measured, mobile phase is methanol-water-glacial acetic acid (15:85:1), detect wavelength 330nm, number of theoretical plate calculates by the chlorogenic acid peak should be not less than 1000, has in the test sample chromatogram and the corresponding to chromatographic peak of the retention time of reference substance.
Cinnamic acid content is 40-51 μ g/mL in the 'Mailuoning ' injection of the present invention, and chlorogenic acid content is 140-205 μ g/mL, and ecdysterone content is 45-50 μ g/mL.
'Mailuoning ' injection of the present invention is characterized in that, in the described 'Mailuoning ' injection intermediate, and content of beary metal≤10ppm, arsenic salt≤1ppm, residue on ignition≤1.5% (g/mL), total solid 13.5-25.5mg/mL; Preferably, residue on ignition≤0.8% (g/mL), total solid 〉=16.3mg/mL.
'Mailuoning ' injection of the present invention is characterized in that, the protein of described 'Mailuoning ' injection intermediate, tannin, resin, oxalates, potassium ion meet the pertinent regulations under 2000 editions injection items of Chinese Pharmacopoeia.
Behind effective component extracting, the injection that the present invention makes can be injection or injectable powder.
During injectable powder, can add various caffolding agents as required in preparation, for example glucose, mannitol, sodium chloride, dextran, sucrose, lactose, gelatin etc., dosage control is at the 1-50% of solution weight.
In the preparation method provided by the invention, adopt the method for alcohol extraction to obtain effective ingredient, after adding aqueous gelatin solution or ZTC clarifier are handled, carry out precipitate with ethanol and filter, purification processes obtains end product.This method technology is simple, and used preparation is easy to obtain, and Impurity removals such as tannin are thorough, and the product yield and the purity that obtain are higher.
" MAILUONING " of the present invention injectable powder contains the raw material components of following weight portion:
Radix Achyranthis Bidentatae 100-1000 Radix Scrophulariae 100-1000
Herba Dendrobii 100-1000 Flos Lonicerae 100-1000
Described weight portion can be measurement units commonly used such as g, kg, two, jin.
The inventive method can realize as follows:
1) Radix Achyranthis Bidentatae in the said components, Radix Scrophulariae, Herba Dendrobii are rinsed well, cut into slices or section or powder, drop in the extraction pot in the lump with Flos Lonicerae, adding the 0%-99.5% ethanol water that 1-10 doubly measures soaked after 1 hour, 40-85 ℃ of following heating and refluxing extraction 1-5 time, the each extraction 1-2 hour filters filtrate for later use; Merging filtrate, being concentrated into relative density is 1.10-1.20 (80 ℃);
2) add 95% ethanol, stir, make the supernatant ethanol content at 75-85%, 0-10 ℃ left standstill 12-24 hour, the leaching supernatant, and reclaiming ethanol and being concentrated into relative density is 1.10-1.20 (80 ℃);
3) ethyl acetate of 1-3 times of concentrated solution volume of adding extracts 3-7 time, extracts 30-60 minute at every turn, merges supernatant, reclaims ethyl acetate, and removes ethyl acetate, is concentrated into thick paste (80 ℃), is effective ingredient.
As prepare lyophilized injectable powder, then add 1-10 and doubly measure water for injection to thick paste, stir, cold preservation 12-24 hour, filter, filtrate adds the poloxamer of 0.1-0.6%, stirring and dissolving, the sodium chloride of adding 0.8-0.9%, stirring and dissolving, transferring PH with 10-40%NaOH solution is between the 7-9, replenish water for injection, add the caffolding agent of solution weight 1-50%, use 0.22-0.45um microporous filter membrane fine straining, the filtrate that obtains fill under aseptic condition is cooled to 0 ℃ in cillin bottle or ampoule, put into freeze drying box then, make the freeze drying box temperature reduce to-40～-60 ℃ rapidly, the messenger drug liquid cooling but, kept 1-5 hour, it is freezed fully; After medicinal liquid freezes fully, the freeze drying box internal pressure is reduced to 1.33～13.33Pa, keeping lyophilizing the temperature inside the box simultaneously is-60～-40 ℃, satisfies the sublimation condition of water, makes the moisture in the medicinal liquid become steam; Be communicated with condenser, make its condensing tube cooling, minimumly can make steam frosting on condensing tube to below-60 ℃, kept 1-10 hour, the freeze drying box shelf slowly is heated to 20-40 ℃ therebetween; Promptly get dried product, goods are taken out seal then.
As prepare injection, and then add additives, according to art methods, be prepared.
Above-mentioned steps 3) also can be to use macroporous resin to carry out purification.
Said method also can be in step 1) and 2) between increase by a step and be: the concentration that adds 1/10 volume in concentrated solution is the 0.5%-10% aqueous gelatin solution, stirs, and places 12-24 hour for 0-10 ℃, and is centrifugal, supernatant.
The inventive method also can be:
1) Radix Achyranthis Bidentatae in the said components, Radix Scrophulariae, Herba Dendrobii are rinsed well, cut into slices or section or powder, drop in the extraction pot in the lump with Flos Lonicerae, adding the 0%-99.5% ethanol water that 1-10 doubly measures soaked after 1 hour, 40-85 ℃ of following heating and refluxing extraction 1-5 time, the each extraction 1-2 hour filters filtrate for later use; Merging filtrate, being concentrated into relative density is 1.10-1.20 (80 ℃);
2) concentration of adding 1/10 volume is the 0.5%-10% aqueous gelatin solution in concentrated solution, stirs, and places 12-24 hour for 0-10 ℃, and is centrifugal, gets supernatant;
3) supernatant is carried out purification by macroporous resin, obtain effective ingredient, be equipped with adjuvant and prepare injectable powder or injection.
The inventive method also can be:
1) Radix Achyranthis Bidentatae in the said components, Radix Scrophulariae, Herba Dendrobii are rinsed well, cut into slices or section or powder, drop in the extraction pot in the lump with Flos Lonicerae, add water logging bubble that 1-10 doubly measures after 1 hour, 40-85 ℃ of following heating and refluxing extraction 1-5 time extracted 1-2 hour at every turn, filter filtrate for later use; Merging filtrate, being concentrated into relative density is 1.10-1.20 (80 ℃);
2) in concentrated solution, add ZTC pretreating agent A component, boil 15-20 minute after, left standstill 4-5 hour, centrifugalize gets the supernatant; Add ZTC pretreating agent A component again, boil 15-20 minute after, left standstill 3 hours, filter, add ZTC clarifier B component in the filtrate, be heated to and boil, placed 4-6 hour, centrifugal after-filtration, filtrate are concentrated into proportion 1.10-1.20 (80 ℃) behind multilamellar filter paper filter press;
3) add 95% ethanol, stir, make the supernatant ethanol content at 75-85%, 0-10 ℃ left standstill 12-24 hour, the leaching supernatant, and reclaiming ethanol and being concentrated into relative density is 1.10-1.20 (80 ℃);
4) carry out purification by macroporous resin, obtain effective ingredient, be equipped with adjuvant and prepare injectable powder or injection.
CN92107848.X compares with Chinese patent, the inventive method helps the extraction fully of effective ingredient, the impurity-removing method that adopts is beneficial to water-solubility impurity and removes fully, make the MAILUONING drug effect obviously promote, the Chinese medicine preparation that uses the inventive method to obtain is greatly improved on purity than the product that traditional method obtains, and has effectively removed tannin; And, adopt injectable powder to replace traditional liquid injection, effectively prolonged the effect duration of medicine, make it have good stability, drug effect can reach more than 3 years.
The specific embodiment:
Embodiment 1-1
Herba Dendrobii, Radix Scrophulariae, each 500g of Radix Achyranthis Bidentatae are cleaned section, and 500g drops into extraction pot with Flos Lonicerae, and 30% ethanol water that adds 6 times of amounts soaked after 1 hour, and 50 ℃ of following heating and refluxing extraction 3 times were extracted 1 hour at every turn, filtered filtrate for later use; Merging filtrate, reclaiming ethanol and being concentrated into relative density is 1.15 (80 ℃);
The concentration that adds 1/10 volume in concentrated solution is 5% gelatin solution, stirs, and places 12 hours for 4 ℃, and is centrifugal, gets supernatant;
In supernatant, add 95% ethanol, stir, the supernatant ethanol content was left standstill 12 hours at 85%, 4 ℃, the leaching supernatant, reclaiming ethanol and being concentrated into relative density is 1.15 (80 ℃);
Add the ethyl acetate of 1 times of concentrated solution volume, extract 3 times, extracted 15 minutes at every turn, merge supernatant, reclaim ethyl acetate, and remove ethyl acetate, be concentrated into thick paste (80 ℃).
Add 3 times of amount waters for injection, stir cold preservation 12 hours, filter, filtrate adds 0.1% poloxamer, stirring and dissolving, the sodium chloride of adding 0.8%, stirring and dissolving, transferring PH with 20%NaOH solution is 8.5, replenish water for injection, the mannitol of adding 1% behind the mix homogeneously, uses 0.45um microporous filter membrane fine straining, the filtrate sterilization, fill is in cillin bottle;
Cillin bottle is cooled to 0 ℃, puts into freeze drying box then, make the freeze drying box temperature reduce to-40 ℃ rapidly, the messenger drug liquid cooling was but kept 5 hours, and medicinal liquid is freezed fully; Then the freeze drying box internal pressure is reduced to 10Pa, keep lyophilizing the temperature inside the box simultaneously and be-40 ℃, make the moisture in the medicinal liquid become steam; Be communicated with condenser, make its condensing tube be cooled to-60 ℃, make steam frosting on condensing tube, the freeze drying box shelf slowly is heated to 21 ℃, keeps 10 hours, promptly gets dried product, goods is taken out seal then.
Embodiment 1-2
Herba Dendrobii 100g, Radix Scrophulariae 300g, Radix Achyranthis Bidentatae 700g are cleaned section, drop into extraction pot in the lump with Flos Lonicerae 1000g, 0.5% ethanol water that adds 1 times of amount soaked after 1 hour, and 40 ℃ of following heating and refluxing extraction 2 times were extracted 2 hours at every turn, filtered filtrate for later use; Merging filtrate, reclaiming ethanol and being concentrated into relative density is 1.10 (50 ℃);
The concentration that adds 1/10 volume in concentrated solution is 0.5% gelatin solution, stirs, and places 12 hours for 4 ℃, and is centrifugal, gets supernatant;
In supernatant, add 95% ethanol, stir, the supernatant ethanol content was left standstill 12 hours at 85%, 4 ℃, the leaching supernatant, reclaiming ethanol and being concentrated into relative density is 1.10 (80 ℃);
Add the ethyl acetate of 1 times of concentrated solution volume, extract 3 times, extracted 60 minutes at every turn, merge supernatant, reclaim ethyl acetate, and remove ethyl acetate, be concentrated into thick paste (80 ℃).
Add 3 times of amount waters for injection, stir cold preservation 12 hours, filter, filtrate adds 0.3% poloxamer, stirring and dissolving, the sodium chloride of adding 0.8%, stirring and dissolving, transferring PH with 30%NaOH solution is 8.5, replenish water for injection, the lactose of adding 20%, mix homogeneously uses 0.22um microporous filter membrane fine straining, the filtrate sterilization, fill is in cillin bottle;
Cillin bottle is cooled to 0 ℃, puts into freeze drying box then, make the freeze drying box temperature reduce to-40 ℃ rapidly, the messenger drug liquid cooling was but kept 4.5 hours, and medicinal liquid is freezed fully; Then the freeze drying box internal pressure is reduced to 7Pa, keep lyophilizing the temperature inside the box simultaneously and be-40 ℃, make the moisture in the medicinal liquid become steam; Be communicated with condenser, make its condensing tube be cooled to-65 ℃, make steam frosting on condensing tube, the freeze drying box shelf slowly is heated to 30 ℃, keeps 3 hours, promptly gets dried product, goods is taken out seal then.
Embodiment 1-3
Herba Dendrobii 1000g, Radix Scrophulariae 100g, Radix Achyranthis Bidentatae 300g are cleaned section, drop into extraction pot in the lump with Flos Lonicerae 700g, 60% ethanol water that adds 10 times of amounts soaked after 1 hour, and 85 ℃ of following heating and refluxing extraction 3 times were extracted 1 hour at every turn, filtered filtrate for later use; Merging filtrate, reclaiming ethanol and being concentrated into relative density is 1.20 (50 ℃);
The concentration that adds 1/10 volume in concentrated solution is 2% gelatin solution, stirs, and places 20 hours for 4 ℃, and is centrifugal, gets supernatant;
In supernatant, add 95% ethanol, stir, the supernatant ethanol content was left standstill 20 hours at 80%, 0 ℃, the leaching supernatant, reclaiming ethanol and being concentrated into relative density is 1.20 (80 ℃);
Add the ethyl acetate of 1 times of concentrated solution volume, extract 5 times, extracted 10 minutes at every turn, merge supernatant, reclaim ethyl acetate, and remove ethyl acetate, be concentrated into thick paste (80 ℃).
Add 10 times of amount waters for injection, stir, 4 ℃ of cold preservation 18 hours, filter, filtrate adds 0.4% poloxamer, stirring and dissolving, the sodium chloride of adding 0.8%, stirring and dissolving, transferring PH with 40%NaOH solution is 8.0, replenishes water for injection, add 10% gelatin, use 0.45um microporous filter membrane fine straining, the filtrate sterilization, fill is in cillin bottle;
Cillin bottle is cooled to 0 ℃, puts into freeze drying box then, make the freeze drying box temperature reduce to-58 ℃ rapidly, the messenger drug liquid cooling was but kept 1.5 hours, and medicinal liquid is freezed fully; Then the freeze drying box internal pressure is reduced to 2Pa, keep lyophilizing the temperature inside the box simultaneously and be-58 ℃, make the moisture in the medicinal liquid become steam; Be communicated with condenser, make its condensing tube be cooled to-65 ℃, make steam frosting on condensing tube, the freeze drying box shelf slowly is heated to 25 ℃, keeps 7 hours, promptly gets dried product, goods is taken out seal then.
Embodiment 1-4
Herba Dendrobii 300g, Radix Scrophulariae 700g, Radix Achyranthis Bidentatae 1000g are cleaned section, drop into extraction pot in the lump with Flos Lonicerae 100g, 99.5% ethanol water that adds 3 times of amounts soaked after 1 hour, and 70 ℃ of following heating and refluxing extraction 3 times were extracted 1 hour at every turn, filtered filtrate for later use; Merging filtrate, reclaiming ethanol and being concentrated into relative density is 1.10 (50 ℃);
The concentration that adds 1/10 volume in concentrated solution is 10% gelatin solution, stirs, and places 12 hours for 8 ℃, and is centrifugal, gets supernatant;
In supernatant, add 95% ethanol, stir, the supernatant ethanol content was left standstill 12 hours at 75%, 8 ℃, the leaching supernatant, reclaiming ethanol and being concentrated into relative density is 1.10 (80 ℃);
Add the ethyl acetate of 1 times of concentrated solution volume, extract 7 times, extracted 5 minutes at every turn, merge supernatant, reclaim ethyl acetate, and remove ethyl acetate, be concentrated into thick paste (80 ℃).
Add 6 times of amount waters for injection, stir cold preservation 24 hours, filter, filtrate adds 0.5% poloxamer, stirring and dissolving, the sodium chloride of adding 0.9%, stirring and dissolving, transferring PH with 20%NaOH solution is 8.5, replenishes water for injection, the glucose of adding 15%, use 0.22um microporous filter membrane fine straining, the filtrate sterilization, fill is in cillin bottle;
Cillin bottle is cooled to 0 ℃, puts into freeze drying box then, make the freeze drying box temperature reduce to-40 ℃ rapidly, the messenger drug liquid cooling was but kept 3.5 hours, and medicinal liquid is freezed fully; Then the freeze drying box internal pressure is reduced to 5Pa, keep lyophilizing the temperature inside the box simultaneously and be-40 ℃, make the moisture in the medicinal liquid become steam; Be communicated with condenser, make its condensing tube be cooled to-50 ℃, make steam frosting on condensing tube, the freeze drying box shelf slowly is heated to 30 ℃, keeps 5 hours, promptly gets dried product, goods is taken out seal then.
Embodiment 2-1
Herba Dendrobii, Radix Scrophulariae, each 500g of Radix Achyranthis Bidentatae are cleaned section, drop into extraction pot in the lump, add 12kg30% ethanol indirect with Flos Lonicerae 500g, extract 3 times, each 1 hour, merge behind 3 extracting liquid filterings, be evaporated to 1/5 and concentrate front volume, the aqueous gelatin solution that adds concentrated solution weight 1%, stir evenly, placed 12 hours for 4 ℃, centrifugal, filter, filtrate decompression concentrates 1/2 volume; Last D101 macroporous resin column is colourless with the deionized water eluting to effluent, then with 85% ethanol elution, after eluent concentrates, obtains injection by the well-established law preparation.
Embodiment 2-2
Herba Dendrobii 100g, Radix Scrophulariae 300g, Radix Achyranthis Bidentatae 700g are cleaned section, drop into extraction pot in the lump with Flos Lonicerae 1000g, add 18.9kg50% ethanol indirect, extracted 3 hours, extracting liquid filtering, filtrate decompression is concentrated into 1/4 and concentrates front volume, adds the aqueous gelatin solution of concentrated solution weight 5%, stirs evenly, placed 24 hours for 10 ℃, centrifugal, filter, filtrate decompression concentrates 1/3 volume; Last D101 macroporous resin column is colourless with the deionized water eluting to effluent, then with 85% ethanol elution, after eluent concentrates, obtains injection by the well-established law preparation.
Embodiment 2-3
Herba Dendrobii 1000g, Radix Scrophulariae 100g, Radix Achyranthis Bidentatae 300g are cleaned section, drop into extraction pot in the lump, add 6.3kg40% ethanol indirect with Flos Lonicerae 700g, extract 2 times, extracted 2 hours for the first time, extracted 1 hour for the second time, extracting solution merges after-filtration, filtrate decompression is concentrated into 1/5 and concentrates front volume, adds the aqueous gelatin solution of concentrated solution weight 3%, stirs evenly, placed 20 hours for 8 ℃, centrifugal, filter, filtrate decompression concentrates 1/4 volume; Last D101 macroporous resin column is colourless with the deionized water eluting to effluent, then with 85% ethanol elution, after eluent concentrates, obtains injection by the well-established law preparation.
Embodiment 2-4
Herba Dendrobii 700g, Radix Scrophulariae 1000g, Radix Achyranthis Bidentatae 100g are cleaned section, drop into extraction pot in the lump, add 10.5kg 40% ethanol indirect with Flos Lonicerae 300g, extract 2 times, the each extraction 2 hours, extracting solution merges after-filtration, and filtrate decompression is concentrated into does not have the alcohol flavor, the ZTC clarifier that adds concentrated solution weight 1%, stir evenly, placed 24 hours for 5 ℃, centrifugal, filter, filtrate decompression concentrates 1/2 volume; Last D101 macroporous resin column is colourless with the deionized water eluting to effluent, then with 85% ethanol elution, after eluent concentrates, obtains injection by the well-established law preparation.
Embodiment 2-5
Herba Dendrobii 300g, Radix Scrophulariae 700g, Radix Achyranthis Bidentatae 1000g are cleaned section, drop into extraction pot in the lump, add 16.8kg30% ethanol indirect with Flos Lonicerae 100g, extract 3 times, extracted 2 hours for the first time, extracted 1 hour for the second time, extracted for the third time 1 hour, three times extracting solution merges after-filtration, and filtrate decompression is concentrated into does not have the alcohol flavor, the ZTC clarifier that adds concentrated solution weight 1%, stir evenly, placed 12 hours for 10 ℃, centrifugal, filter, filtrate decompression concentrates 1/3 volume; Last D101 macroporous resin column is colourless with the deionized water eluting to effluent, then with 85% ethanol elution, after eluent concentrates, obtains injection by the well-established law preparation.
Embodiment 2-6
Herba Dendrobii, Radix Scrophulariae, each 500g of Radix Achyranthis Bidentatae are cleaned section, drop into extraction pot in the lump, add 12kg50% ethanol indirect with Flos Lonicerae 500g, extract 3 times, each 1 hour, merge behind 3 extracting liquid filterings, being evaporated to does not have the alcohol flavor, the ZTC clarifier that adds concentrated solution weight 1%, stir evenly, placed 22 hours for 8 ℃, centrifugal, filter, filtrate decompression concentrates 1/4 volume; Last D101 macroporous resin column is colourless with the deionized water eluting to effluent, then with 85% ethanol elution, after eluent concentrates, obtains injection by the well-established law preparation.
Embodiment 3-1
With Herba Dendrobii, Radix Scrophulariae, each 500g of Radix Achyranthis Bidentatae cleans section, drop into decoction pot with Flos Lonicerae 500g, add 12kg water, be heated to boiling, decoct 3 times, each 1 hour, 3 decoction liquor were filtered the back and are merged, and are evaporated to 1/2 and concentrate front volume, the aqueous gelatin solution that adds concentrated solution volume 5%, stir, placed 24 hours down for 10 ℃, centrifugal back filter paper filtering, filtrate decompression is concentrated into 1/4 and concentrates front volume, the ethanol of adding 95% is 80% to containing the alcohol amount, places 12 hours filter paper filtering down at 5 ℃, filtrate is 20% NaOH solution accent PH=8.0 with concentration, placed 12 hours, filter paper filtering, filtrate decompression is concentrated into does not have the alcohol flavor; With the ethyl acetate extraction of 1 times of volume 3 times, reclaim under reduced pressure obtains injection to tasteless by the well-established law preparation.
Embodiment 3-2
With Herba Dendrobii 100g, Radix Scrophulariae 300g, Radix Achyranthis Bidentatae 700g cleans section, drop into decoction pot with Flos Lonicerae 1000g, add 18.9kg water, be heated to boiling, decocted 3 hours, decoction liquor is filtered, filtrate decompression is concentrated into 1/3 and concentrates front volume, adds the aqueous gelatin solution of concentrated solution volume 1%, stirs, placed 12 hours down for 5 ℃, centrifugal back filter paper filtering, filtrate decompression is concentrated into 1/3 and concentrates front volume, and the ethanol of adding 95% is 80% to containing the alcohol amount, placed 24 hours down at 10 ℃, filter paper filtering, filtrate are that 10% NaOH solution is transferred PH=8.5 with concentration, place 24 hours, filter paper filtering, filtrate decompression are concentrated into does not have the alcohol flavor; With the ethyl acetate extraction of 3 times of volumes 1 time, reclaim under reduced pressure obtains injection to tasteless by the well-established law preparation.
Embodiment 3-3
With Herba Dendrobii 1000g, Radix Scrophulariae 100g, Radix Achyranthis Bidentatae 300g cleans section, drop into decoction pot with Flos Lonicerae 700g, add 6.3kg water, be heated to boiling, decoct 2 times, decocted 2 hours for the first time, decocted 1 hour for the second time, decoction liquor merges after-filtration, filtrate decompression is concentrated into 1/2 and concentrates front volume, adds the aqueous gelatin solution of concentrated solution volume 5%, stirs, placed 15 hours down for 8 ℃, centrifugal back filter paper filtering, filtrate decompression is concentrated into 1/4 and concentrates front volume, and the ethanol of adding 95% is 80% to containing the alcohol amount, placed 20 hours down at 8 ℃, filter paper filtering, filtrate are that 30% NaOH solution is transferred PH=7.5 with concentration, place 18 hours, filter paper filtering, filtrate decompression are concentrated into does not have the alcohol flavor; With the ethyl acetate extraction of 2 times of volumes 3 times, reclaim under reduced pressure obtains injection to tasteless by the well-established law preparation.
Embodiment 3-4
With Herba Dendrobii 700g, Radix Scrophulariae 1000g, Radix Achyranthis Bidentatae 100g cleans section, drop into decoction pot with Flos Lonicerae 300g, add 10.5kg water, be heated to boiling, decoct 2 times, the each decoction 2 hours, decoction liquor merges after-filtration, and filtrate decompression is concentrated into 1/3 and concentrates front volume, the aqueous gelatin solution that adds concentrated solution volume 1%, stir, placed 20 hours down for 7 ℃, centrifugal back filter paper filtering, filtrate decompression is concentrated into 1/3 and concentrates front volume, the ethanol of adding 95% is 80% to containing the alcohol amount, places 24 hours filter paper filtering down at 10 ℃, filtrate is 20% NaOH solution accent PH=8.5 with concentration, placed 12 hours, filter paper filtering, filtrate decompression is concentrated into does not have the alcohol flavor; With the ethyl acetate extraction of 2 times of volumes 2 times, reclaim under reduced pressure obtains injection to tasteless by the well-established law preparation.
Embodiment 3-5
With Herba Dendrobii 300g, Radix Scrophulariae 700g, Radix Achyranthis Bidentatae 1000g cleans section, drop into decoction pot with Flos Lonicerae 100g, add 16.8kg water, be heated to boiling, decoct 3 times, decocted 2 hours for the first time, decocted 1 hour for the second time, decocted for the third time 1 hour, three times decoction liquor merges after-filtration, and filtrate decompression is concentrated into 1/2 and concentrates front volume, the aqueous gelatin solution that adds concentrated solution volume 3%, stir, placed 12 hours down for 5 ℃, centrifugal back filter paper filtering, filtrate decompression is concentrated into 1/4 and concentrates front volume, the ethanol of adding 95% is 80% to containing the alcohol amount, places 12 hours filter paper filtering down at 5 ℃, filtrate is 20% NaOH solution accent PH=8.0 with concentration, placed 24 hours, filter paper filtering, filtrate decompression is concentrated into does not have the alcohol flavor; With the ethyl acetate extraction of 3 times of volumes 3 times, reclaim under reduced pressure obtains injection to tasteless by the well-established law preparation.
Embodiment 3-6
With Herba Dendrobii, Radix Scrophulariae, each 500g of Radix Achyranthis Bidentatae cleans section, drop into decoction pot with Flos Lonicerae 500g, add 12kg water, be heated to boiling, decoct 3 times, each 1 hour, 3 decoction liquor were filtered the back and are merged, and are evaporated to 1/2 and concentrate front volume, the aqueous gelatin solution that adds concentrated solution volume 5%, stir, placed 24 hours down for 10 ℃, centrifugal back filter paper filtering, filtrate decompression is concentrated into 1/4 and concentrates front volume, the ethanol of adding 95% is 80% to containing the alcohol amount, places 12 hours filter paper filtering down at 5 ℃, filtrate is 20% NaOH solution accent PH=8.0 with concentration, placed 12 hours, filter paper filtering, filtrate decompression is concentrated into does not have the alcohol flavor; With the ethyl acetate extraction of 3 times of volumes 2 times, reclaim under reduced pressure obtains injection to tasteless by the well-established law preparation.
Embodiment 4-1
Herba Dendrobii, Radix Scrophulariae, each 500g of Radix Achyranthis Bidentatae are cleaned section, drop into decoction pot with Flos Lonicerae 500g, add 12kg water, be heated to boiling, decoct each 1 hour 3 times, 3 decoction liquor are filtered the back and are merged, be evaporated to 1/2 and concentrate front volume, under 60 ℃, add the ZTC clarifier of concentrated solution weight 1%, placed 12 hours down at 5 ℃, centrifugal, to filter, filtrate decompression is concentrated into 1/4 and concentrates front volume, adding 95% ethanol then is 80% to containing the alcohol amount, placed 24 hours for 10 ℃, filter, filtrate decompression is concentrated into does not have the alcohol flavor; Last D101 macroporous resin column is colourless with the deionized water eluting to effluent, then with 85% ethanol elution, after eluent concentrates, obtains injection by the well-established law preparation.
Embodiment 4-2
Herba Dendrobii 100g, Radix Scrophulariae 300g, Radix Achyranthis Bidentatae 700g are cleaned section, drop into decoction pot with Flos Lonicerae 1000g, add 18.9kg water, be heated to boiling, decocted 3 hours, decoction liquor is filtered, and filtrate decompression is concentrated into 1/3 and concentrates front volume, under 55 ℃, the ZTC clarifier that adds concentrated solution weight 5% is placed down at 10 ℃, 24 hours, and is centrifugal, filter, filtrate decompression is concentrated into 1/3 and concentrates front volume, adds 95% ethanol then and is 80%, 5 ℃ and placed 12 hours to containing the alcohol amount, filter, filtrate decompression is concentrated into does not have the alcohol flavor; Last D101 macroporous resin column is colourless with the deionized water eluting to effluent, then with 85% ethanol elution, after eluent concentrates, obtains injection by the well-established law preparation.
Embodiment 4-3
Herba Dendrobii 1000g, Radix Scrophulariae 100g, Radix Achyranthis Bidentatae 300g are cleaned section, drop into decoction pot with Flos Lonicerae 700g, add 6.3kg water, be heated to boiling, decoct 2 times, decocted 2 hours for the first time, decocted 1 hour for the second time, decoction liquor merges after-filtration, and filtrate decompression is concentrated into 1/2 and concentrates front volume, under 65 ℃, the ZTC clarifier that adds concentrated solution weight 3% was placed 20 hours down at 8 ℃, and is centrifugal, filter, filtrate decompression is concentrated into 1/3 and concentrates front volume, adds 95% ethanol then and is 80%, 10 ℃ and placed 18 hours to containing the alcohol amount, filter, filtrate decompression is concentrated into does not have the alcohol flavor; Last D101 macroporous resin column is colourless with the deionized water eluting to effluent, then with 85% ethanol elution, after eluent concentrates, obtains injection by the well-established law preparation.
Embodiment 5-1
With Herba Dendrobii 700g, Radix Scrophulariae 1000g, Radix Achyranthis Bidentatae 100g cleans section, drop into decoction pot with Flos Lonicerae 300g, add 10.5kg water, be heated to boiling, decoct 2 times, the each decoction 2 hours, decoction liquor merges after-filtration, and filtrate decompression is concentrated into 1/3 and concentrates front volume, adds the aqueous gelatin solution of concentrated solution weight 5%, stir, placed 15 hours down at 5 ℃, centrifugal, filter, filtrate decompression is concentrated into 1/4 and concentrates front volume, add 95% ethanol then and be 80%, 8 ℃ and placed 24 hours, filter to containing the alcohol amount, after filtrate transferred pH value to be 8.0 with 20% NaOH solution, being evaporated to did not have the alcohol flavor; Last D101 macroporous resin column is colourless with the deionized water eluting to effluent, then with 85% ethanol elution, after eluent concentrates, obtains injection by the well-established law preparation.
Embodiment 5-2
With Herba Dendrobii 300g, Radix Scrophulariae 700g, Radix Achyranthis Bidentatae 1000g cleans section, drop into decoction pot with Flos Lonicerae 100g, add 16.8kg water, be heated to boiling, decoct 3 times, decocted 2 hours for the first time, decocted 1 hour for the second time, decocted for the third time 1 hour, three times decoction liquor merges after-filtration, and filtrate decompression is concentrated into 1/2 and concentrates front volume, the aqueous gelatin solution that adds concentrated solution weight 1%, stir, placed 12 hours down at 10 ℃, centrifugal, filter, filtrate decompression is concentrated into 1/3 and concentrates front volume, adds 95% ethanol then and is 80%, 10 ℃ and placed 12 hours to containing the alcohol amount, filter, it is that placed 12 hours 8.5 backs that filtrate is transferred pH value with 20% NaOH solution, filters, and filtrate decompression is concentrated into does not have the alcohol flavor; Last D101 macroporous resin column is colourless with the deionized water eluting to effluent, then with 85% ethanol elution, after eluent concentrates, obtains injection by the well-established law preparation.
Embodiment 5-3
With Herba Dendrobii, Radix Scrophulariae, each 500g of Radix Achyranthis Bidentatae cleans section, drops into decoction pot with Flos Lonicerae 500g, adds 12kg water, be heated to boiling, decoct 3 times, each 1 hour, 3 decoction liquor were filtered the back and are merged, be evaporated to 1/2 and concentrate front volume, add the aqueous gelatin solution of concentrated solution weight 3%, stir, placed 12 hours down at 8 ℃, centrifugal, filter, filtrate decompression is concentrated into 1/4 and concentrates front volume, and adding 95% ethanol then is 80% to containing the alcohol amount, placed 22 hours for 10 ℃, filter, filtrate was placed 12 hours after transferring pH value to be 8.5 with 20% NaOH solution, filter, filtrate decompression is concentrated into does not have the alcohol flavor; Last D101 macroporous resin column is colourless with the deionized water eluting to effluent, then with 85% ethanol elution, after eluent concentrates, obtains injection by the well-established law preparation.
Embodiment 6-1---6-3
After removing the decoction liquor concentrating under reduced pressure, do not add aqueous gelatin solution, directly adding 95% ethanol is beyond 80% the step to containing the alcohol amount, and all the other steps are with embodiment 5-1,5-2,5-3.
Embodiment 7-1
Herba Dendrobii, Radix Scrophulariae, each 500g of Radix Achyranthis Bidentatae are cleaned section, drop into decoction pot, add 30% ethanol of 12kg with Flos Lonicerae 500g, be heated to boiling, decoct each 1 hour 3 times, 3 decoction liquor are filtered the back and are merged, and are evaporated to 1/2 and concentrate front volume, and the ethanol of adding 95% is 80% to containing the alcohol amount, placed 12 hours down at 5 ℃, filter paper filtering, filtrate are that 20% NaOH solution is transferred PH=8.0 with concentration, place 12 hours, filter paper filtering, filtrate decompression are concentrated into does not have the alcohol flavor; Last D101 macroporous resin column is colourless with the deionized water eluting to effluent, then with 85% ethanol elution, after eluent concentrates, obtains injection by the well-established law preparation.
Embodiment 7-2
Herba Dendrobii 100g, Radix Scrophulariae 300g, Radix Achyranthis Bidentatae 700g are cleaned section, drop into decoction pot, add 40% ethanol of 18.9kg with Flos Lonicerae 1000g, be heated to boiling, decocted 3 hours, decoction liquor is filtered, filtrate decompression is concentrated into 1/3 and concentrates front volume, the ethanol of adding 95% is 80% to containing the alcohol amount, places 24 hours filter paper filtering down at 10 ℃, filtrate is 10% NaOH solution accent PH=8.5 with concentration, placed 24 hours, filter paper filtering, filtrate decompression is concentrated into does not have the alcohol flavor; Last D101 macroporous resin column is colourless with the deionized water eluting to effluent, then with 85% ethanol elution, after eluent concentrates, obtains injection by the well-established law preparation.
Embodiment 7-3
Herba Dendrobii 1000g, Radix Scrophulariae 100g, Radix Achyranthis Bidentatae 300g are cleaned section, drop into decoction pot with Flos Lonicerae 700g, 50% ethanol that adds 6.3kg, be heated to boiling, decoct 2 times, decocted 2 hours for the first time, decocted 1 hour for the second time, decoction liquor merges after-filtration, filtrate decompression is concentrated into 1/4 and concentrates front volume, the ethanol of adding 95% is 80% to containing the alcohol amount, places 20 hours filter paper filtering down at 8 ℃, filtrate is 30% NaOH solution accent PH=7.5 with concentration, placed 18 hours, filter paper filtering, filtrate decompression is concentrated into does not have the alcohol flavor; Last D101 macroporous resin column is colourless with the deionized water eluting to effluent, then with 85% ethanol elution, after eluent concentrates, obtains injection by the well-established law preparation.
Embodiment 7-4
Herba Dendrobii 700g, Radix Scrophulariae 1000g, Radix Achyranthis Bidentatae 100g are cleaned section, drop into extraction pot, add 10.5kg30% ethanol with Flos Lonicerae 300g, indirect is extracted 2 times to boiling, extracts 2 hours at every turn, extracting solution merges after-filtration, and filtrate decompression is concentrated into 1/3 and concentrates front volume, and the ethanol of adding 95% is 80% to containing the alcohol amount, placed 24 hours down at 10 ℃, filter paper filtering, filtrate are that 20% NaOH solution is transferred PH=8.5 with concentration, place 12 hours, filter paper filtering, filtrate decompression are concentrated into does not have the alcohol flavor; Last D101 macroporous resin column is colourless with the deionized water eluting to effluent, then with 85% ethanol elution, after eluent concentrates, obtains injection by the well-established law preparation.
Embodiment 7-5
Herba Dendrobii 300g, Radix Scrophulariae 700g, Radix Achyranthis Bidentatae 1000g are cleaned section, drop into extraction pot with Flos Lonicerae 100g, add 16.8kg40% ethanol, indirect is to boiling, extract 3 times, extracted 2 hours for the first time, extracted 1 hour for the second time, extracted for the third time 1 hour, three times extracting solution merges after-filtration, and filtrate decompression is concentrated into 1/2 and concentrates front volume, and the ethanol of adding 95% is 80% to containing the alcohol amount, placed 12 hours down at 5 ℃, filter paper filtering, filtrate are that 20% NaOH solution is transferred PH=8.0 with concentration, place 24 hours, filter paper filtering, filtrate decompression are concentrated into does not have the alcohol flavor; Last D101 macroporous resin column is colourless with the deionized water eluting to effluent, then with 85% ethanol elution, after eluent concentrates, obtains injection by the well-established law preparation.
Embodiment 7-6
Herba Dendrobii, Radix Scrophulariae, each 500g of Radix Achyranthis Bidentatae are cleaned section, drop into decoction pot, add 12kg50% ethanol with Flos Lonicerae 500g, indirect is extracted each 1 hour 3 times to boiling, merge behind 3 extracting liquid filterings, be evaporated to 1/2 and concentrate front volume, the ethanol of adding 95% is 80% to containing the alcohol amount, placed 12 hours down at 5 ℃, filter paper filtering, filtrate are that 20% NaOH solution is transferred PH=8.0 with concentration, place 12 hours, filter paper filtering, filtrate decompression are concentrated into does not have the alcohol flavor; Last D101 macroporous resin column is colourless with the deionized water eluting to effluent, then with 85% ethanol elution, after eluent concentrates, obtains injection by the well-established law preparation.
Comparative example 1
This example is the assay of Herba Dendrobii in the MAILUONING ZHUSHEYE:
1. instrument and reagent
Waters 2695 high performance liquid chromatographs; Waters 2996 diode array detector; The Empower data processing software;
Reagent:
Acetonitrile (chromatographically pure): Fisher Scientific company, lot number: 020109
Water: double distilled water;
The escoparone reference substance: Nat'l Pharmaceutical ﹠ Biological Products Control Institute provides, for assay usefulness, lot number: 1511-200001.
2. chromatographic condition
Chromatographic column: Kromasil C18 (Φ 4.6 * 200mm, 5 μ m)
Column temperature: 30 ℃
Mobile phase: acetonitrile-water (1:4), flow velocity: 0.8ml/min
Sample size: 10 μ l
Wavelength: 343nm
3. sample preparation
It is an amount of that the preparation precision of reference substance solution takes by weighing the escoparone reference substance, adds methanol and make the solution that contains 5 μ g among every 1ml, promptly.
Need testing solution: get product of the present invention, add the injection water, make the 50ml MAILUONING ZHUSHEYE, add sodium chloride 5g, use chloroform extraction 3 times, each 30ml, combined chloroform liquid, put evaporate to dryness in the water-bath, residue adds 60% methanol makes dissolving, and is transferred in the 25ml measuring bottle, add 60% methanol and be diluted to scale, shake up, filter with 0.45 μ m microporous filter membrane, filtrate is as need testing solution.
The negative control sample that lacks Herba Dendrobii: take by weighing Radix Scrophulariae, Radix Achyranthis Bidentatae, each 50g of Flos Lonicerae,, make the MAILUONING ZHUSHEYE that lacks Herba Dendrobii, make the negative control solution that lacks Herba Dendrobii according to the preparation method of above-mentioned need testing solution according to the operation of embodiment 1-1.
4. negative control test
Under aforementioned chromatographic condition, the accurate negative control solution 10 μ l that draw above-mentioned reference substance solution and lack Herba Dendrobii, inject chromatograph of liquid, record HPLC figure, the result shows that (about 21.4 minutes) no chromatographic peak occurs negative sample at retention time place, escoparone peak, illustrates that negative sample is noiseless to the mensuration of escoparone in the sample.
5. chromatographic isolation effect
Get need testing solution 10 μ l sample introduction under above-mentioned chromatographic condition and measure, in the gained chromatogram, the number of theoretical plate at escoparone peak is 5922, and separates well with adjacent chromatographic peak, and separating degree is 3.5.
6. finished product assay and result
By preceding method five batches of finished products are measured, measurement result sees the following form:
| The content meansigma methods of escoparone (μ g/ml) | |
| Embodiment 1-1 | 4.26 |
| Embodiment 2-1 | 4.40 |
| Embodiment 3-1 | 3.90 |
| Embodiment 4-1 | 3.48 |
| Embodiment 5-1 | 3.26 |
| Meansigma methods | 3.86 |
The escoparone measurement result of the MAILUONING ZHUSHEYE sample of the explained hereafter of describing according to Chinese patent CN92107848.X sees the following form:
| Lot number | Escoparone content meansigma methods (μ g/ml) |
| 1 | 2.05 |
| 2 | 2.80 |
| 3 | 2.57 |
| 4 | 2.11 |
| 5 | 2.65 |
| Meansigma methods | 2.44 |
7. conclusion
Five batches of sample escoparone content meansigma methodss of producing by new technology are 3.86 μ g/ml.
The escoparone content meansigma methods of the MAILUONING ZHUSHEYE sample of the explained hereafter of describing according to Chinese patent CN92107848.X is 2.44 μ g/ml.
Therefore the product of the inventive method production is compared with the technology that Chinese patent CN92107848.X describes, and the escoparone content of the product of producing significantly improves.
Comparative example 3
This example contrasts for the quality of the product that the inventive method and Chinese patent CN92107848.X technology make.Two handicraft product quality contrast tables:
Conclusion:
The composition that process of the present invention can be extracted Chinese patent CN92107848.X all extracts, and every index of the product of being produced is better than the sample that Chinese patent CN92107848.X produces.
Comparative example 4
This example compares for the animal experimental data of product of the present invention.
Test material:
1 is tried thing:
Title: MAILUONING extract injection
Specification: every bottle of 100mL (every mL contains the suitable crude drug amount of MAILUONING extract 20g)
The unit of providing: Harbin work of nature academy of science
Lot number: 030211
2 experimental animals:
2.1 white mice Kunming kind is male, body weight 18-22g, 28-32g.
2.2 rat SD is male, body weight 300-350g.
2.3 rabbit New Zealand plants, and white rabbit is male, the above animal of body weight 2.0-3.0kg. provides by Shenyang Pharmaceutical University's Experimental Animal Center, the quality certification number: SYXK (the Liao Dynasty) 2003-008
3 contrast medicines:
3.1 MAILUONING ZHUSHEYE: Jinling Pharmaceutical Co., Ltd., Jinling Pharmaceutical Factory, lot number: 20030305.
3.2 adenosine diphosphate (ADP) disodium salt: Shanghai chemical reagents corporation of Chinese Medicine group, lot number: 20030407
3.3 thrombin: biochemical-pharmaceutical factory, the first biochemical Pharma Inc. Shanghai, Shanghai, lot number: 021015.
3.4 collagen: press the literature method self-control.
3.5 macromolecule right rotary glycoside (Dextron 400): molecular weight is 400,000, Shenyang No. 1 Pharmaceutical Factory production.
3.6 poloxamer: 188 models (F68) polymer, fine chemical product portion of Shanghai branch company of Basf AG.
4. key instrument
4.1LG-KOALA platelet aggregation tester Beijing Steellex Scientific Instrument Company
4.2 autobalance micro centrifuge LDZ4-0.8 Beijing Medical Centrifugal Machine Factory
4.3 N5A-type Puli hemopoietic fluid viscosity meter
4.4 the vast scientific instrument company limited in S501-A type water bath with thermostatic control Liaoyang
4.5 micro blood centrifuge: SH-120, Shanghai Surgical Operation Equipment Factory
4.6 centrifugation device: 80-2, Shanghai Surgical Operation Equipment Factory
Test method and result:
1. the MAILUONING extract injection is to the influence of rat platelet aggregation
1.1 influence to the inductive rat platelet aggregation of adenosine diphosphate (ADP)
Animal subject is divided into 6 groups by the body weight equilibrium, be respectively solvent control group, 3 dosage groups of MAILUONING intravenously administrable, dosage is respectively 40g/kg, 20g/kg, 10g/kg, and other establishes MAILUONING gastric infusion group, dosage is 20g/kg and commercially available MAILUONING intravenously administrable group, and dosage is 20g/kg.The matched group vein gives 1% poloxamer normal saline, the administration volume is 5mL/kg, administration every day 1 time, continuous 7 days, 30min socket of the eye vein was got blood 2mL after the last administration, with 3.8% sodium citrate (1:9) anticoagulant, conventional preparation PRP and PPP, add the 10uL1.06uM adenosine diphosphate (ADP), on LG-KOALA platelet aggregation tester, measure and respectively organize platelet aggregation rate, and obtain the gathering suppression ratio.
Assemble suppression ratio %=(matched group is assembled %-administration group and assembled %)/matched group and assemble % * 100%
With the aggregation rate is to compare significance test between index is organized.
Result of the test is as shown in the table, and MAILUONING ZHUSHEYE 10,20,40g/kg have the inhibitory action of highly significant to the inductive rat platelet aggregation of ADP, and its effect obviously is better than the oral administration group, the commercially available MAILUONING group of dosage such as is better than.
The MAILUONING intravenous administration to the influence of the inductive rat platelet aggregation of ADP (n=10, x ± s) see the following form:
Compare with matched group
*P＜0.05,
*Compare with oral MAILUONING group p＜0.01
++P＜0.01
1.2 influence to collagen-induced rat platelet aggregation
Tried rat grouping, administration and got the blood measuring method with " 1.1 ", aggregating agent prepared therefrom changes 20uL collagen (by the 2nd edition method self-control of uncle Xu cloud pharmacological experimental methodology) into, the gathering time changes 10min into, data statistics is with " 1.1 ", shown in result of the test sees the following form, MAILUONING ZHUSHEYE 10,20,40g/kg have the inhibitory action of highly significant to collagen-induced rat platelet aggregation, and its effect obviously is better than the oral administration group, the commercially available MAILUONING group of dosage such as are better than.
The MAILUONING intravenous administration to the influence of collagen-induced rat platelet aggregation (n=10, x ± s) see the following form:
Compare with matched group
*P＜0.05,
*P＜0.01,
Compare with oral MAILUONING group
++P＜0.01
1.3 influence to the rat platelet aggregation of thrombin induction
Tried rat grouping, administration and got the blood measuring method with " 1.1 ", aggregating agent prepared therefrom changes the thrombin of 10uL2u/mL into, data statistics is with " 1.1 ", shown in result of the test sees the following form, MAILUONING ZHUSHEYE 10,20,40g/kg have the inhibitory action of highly significant to the rat platelet aggregation of thrombin induction, and its effect obviously is better than the oral administration group.
Table 3. MAILUONING intravenous administration is to the influence of the rat platelet aggregation of thrombin induction (n=10, x ± s)
Compare with matched group
*P＜0.05,
*Compare with oral MAILUONING group p＜0.01
++P＜0.01
2. the MAILUONING extract injection is to the thrombotic influence of platelet dependency:
2.1 to the thrombotic influence of rabbit common carotid artery:
Animal subject divides 6 groups, is respectively matched group, 3 dosage groups of MAILUONING intravenously administrable are respectively 20g/kg, 10g/kg, 5g/kg, and other establishes MAILUONING gastric infusion group, and dosage is 10g/kg and commercially available MAILUONING intravenously administrable group, and dosage is 10g/kg.The matched group vein is given and 1% poloxamer normal saline, the administration volume is 5mL/kg, be administered once every day, continuous 7 days, after the last administration, use pentobarbital sodium (about 30mg/kg) intravenous anesthesia, back of the body position is fixing, a free side common carotid artery, at a distance of the 4cm place tremulous pulse two ends are being clamped with bulldog clamp, to be about the 3cm4 suture silk with No. 12 thin sewing-needles sews up in the common carotid artery, open bulldog clamp, recover blood flow (above-mentioned work in the last administration after 0.5h finish), behind the 2h common carotid artery is cut, cut off blood vessel, remove surplus blood and extract silk thread out with the filter paper suction, claim every group every Sanguis Leporis seu oryctolagi bolt weight in wet base, and calculate thrombosis suppression ratio (thrombosis suppression ratio=(matched group wet weight of thrombus-administration group wet weight of thrombus)/matched group wet weight of thrombus * 100%
Result of the test is as shown in the table, and in this selective agent weight range, MAILUONING ZHUSHEYE is formed with the highly significant inhibitory action to platelet thrombus, and MAILUONING ZHUSHEYE is better than waiting commercially available contrast medicine of dosage and gastric infusion group.
The MAILUONING intravenous administration to the thrombotic influence of rabbit common carotid artery (X ± SD) see the following form:
Each administration group and matched group compare,
*P＜0.05,
*P＜0.01.
2.2 protective effect to the mouse lung thromboembolism
Get 120 of mices (male); by 2.1 groupings; every group 20; administration group dosage is respectively 15g/kg; 30g/kg; 60g/kg (iv) establishes ig administration group in addition; dosage is 30g/kg and commercially available MAILUONING ZHUSHEYE 30g/kg group; the administration volume is 0.2mL/10g; injection speed is 0.05mL/s; successive administration 7 days; 0.5 hour mouse tail vein injects collagen (Sigma company production after the last administration; c-8886, lot108e8015) with the strong co-induction agent of epinephrine 5.0mL/kg (collagen 2.5mg/kg+ epinephrine 2mg/kg), injection speed is 0.1mL/5s; observe dead mouse situation in the 3min; with every group of dead animal number and protective rate is observation index, and result of the test is as shown in the table, through x
2Check shows that in selected dosage range the MAILUONING ZHUSHEYE high dose group causes death to collagen and epinephrine inducing mouse lung thrombosis remarkable protective effect is arranged.Tried the thing effect and be better than commercially available MAILUONING, be better than same dose gastric infusion group.MAILUONING ZHUSHEYE is brought out the protective effect of mouse lung thrombosis to collagen+epinephrine complex and is seen the following form:
2.3 the blocking-up middle cerebral artery is caused the influence of focal cerebral ischemia
70 male rats are divided into 7 groups at random, 10 every group, are respectively pseudo-operation group, model control group, 3 dosage groups of MAILUONING ZHUSHEYE dosage and are respectively 10g/kg, 20g/kg, 40g/kg; MAILUONING ZHUSHEYE gastric infusion group dosage is 20g/kg, commercially available MAILUONING 20g/kg intravenous administration group, pseudo-operation group and model control group intravenous injection 1% poloxamer normal saline, administration every day 1 time, successive administration 7 days, administration volume are 0.5mL/100g, and injection speed is 2mL/min, face and use preceding preparation, it is identical that each organizes the administration volume.After the last administration 30min with 10% chloral hydrate with the 350mg/kg intraperitoneal injection of anesthesia, it is fixing to be tried rat back of the body position, the operation of throat portion, separate right carotid to arteria pterygopalatina and ligation it, with diameter is that 0.24mm import fishing line penetrates in common carotid artery and internal carotid artery bifurcation otch, penetrate the about 18mm of length, layer-by-layer suture then, postoperative was observed and is checked scoring one by one by disordered brain function index standards of grading (seeing attached list) in 24 hours, as the disordered brain function evaluation index, result of the test such as following table compare with model control group, and each group of MAILUONING ZHUSHEYE is to the disordered brain function effect of being significantly improved.
After scoring finished, the sacrificed by decapitation rat was got brain and washes with cold saline, took out after putting into-20 ℃ of freezing 5min of refrigerator, removed olfactory bulb, cerebellum and low brain stem, was cut into 5 along coronalplane, and the brain sheet is put into 5mLTTC dyeing (4%TTC1.5mL, 1mol/LK
2HPO
40.1mL and 3.4mL distilled water), 37 ℃ of lucifuge 30min, normal cerebral tissue takes on a red color, infarcted region is because of painted being white in color not, brain weight before the weighing infarcted region reaches respectively, before accounting for infarcted region the percentage ratio of brain weight be between observation index is organized relatively, significance test result such as following table, the percentage ratio of brain weight before MAILUONING ZHUSHEYE significantly reduces the weight of stopping up rat cerebral infarction position due to the middle cerebral artery and infarction position weight and accounts in selected dosage range.Result of the test shows that being tried thing is better than commercially available MAILUONING, is better than same dose gastric infusion group.
MAILUONING ZHUSHEYE to the improvement effect of MCAO rat nervous symptoms (X ± SD) see the following form:
Each administration group and model group compare,
*P＜0.05,
*P＜0.01.
MAILUONING ZHUSHEYE to the protective effect of MCAO rat cerebral infarction (X ± SD) see the following form:
Administration group and matched group compare,
*P＜0.05,
*P＜0.01.
Subordinate list: disordered brain function index
| The disordered brain function symptom | Scoring |
| 1. carry the about chi of Mus tail built on stilts, observe forelimb flexing situation, if two forelimb symmetry is stretched to ground, score is 0; If the flexing of wrist flexing, elbow flexing, shoulder inward turning or existing wrist, elbow appears in operation offside forelimb, receipts person in takeing on is counted 1,2,3 fen respectively and 4 minutes. | 0～4 |
| 2. animal is placed on the plane earth, pushes away both shoulders respectively to side shifting, if bilateral equates and is strong that score is 0, as resistance descender when the operation offside promotes, according to the decline degree be divided into gently, in, weigh 3 and spend, count 1,2,3 fen respectively. | 0～3 |
| 3. animal two forelimbs are put on the wire netting, observed the muscular tension of two forelimbs, both sides muscular tension equity and strong person count 0 fen; Count 1,2,3 fen according to operation offside muscular tension decline degree difference. | 0～3 |
| 4. animal is placed on the plane earth, counted 1 fen to a side person of turn-taking. | 1 |
2.4 to the thrombotic influence of rat postcava:
60 rats are divided into groups by body weight is balanced, every group 10, grouping and administering mode, dosage are with " 2.3 " test, fix with pentobarbital sodium 350mg/kg intraperitoneal injection of anesthesia back position, do median abdominal incision, go deep into the abdominal cavity, separate postcava, after the left renal vein below is with No. 4 silk thread ligation postcava, close abdomen, again open abdomen after 2 hours, 2cm place folder closes blood vessel and cuts tube chamber open, removal of thromboses under ligature, be stained with except that enclosing surperficial blood and claim weight in wet base, calculating wet weight of thrombus and thrombosis suppression ratio.Result of the test is as shown in table 8, and in the selective agent weight range, MAILUONING ZHUSHEYE has remarkable inhibitory action to venous thrombosis, and the intravenously administrable group is better than irritates stomach group and commercially available same dose group.
MAILUONING ZHUSHEYE to the thrombotic influence of rat postcava (X ± SD) see the following form:
Administration group and matched group compare,
*P＜0.05,
*P＜0.01.
3. the MAILUONING extract injection influences microcirculation of mouse auricle
Tried mice with 60 and be divided into 6 groups: be respectively oral dose administration group (30g/kg), commercially available MAILUONING group (30g/kg) in solvent control group (1% poloxamer normal saline), the basic, normal, high dosage group of MAILUONING extract injection (15,30,60g/kg), the MAILUONING by body weight, sex equilibrium, all intravenous injections except that the 5th group, the administration volume is 0.2mL/10g, successive administration 7 days, every day 1 time.Give mice intramuscular injection anesthesia with 20% urethane 0.7mL/100g after the last administration, draw the Mice Auricle hair gently with rubber plaster, the abdomen position is fixed on the observation platform, and auricle is open and flat, and with 10 * 10 times of sem observation mice microcirculations, aforesaid operations is finished in 30min.Numeration auricle blood capillary opening amount is with micro-micrometer pick up the ears wide tremulous pulse (A), vein (V) bore.MAILUONING extract injection 30,60g/kg have improvement effect very significantly to the Mice Auricle microcirculation, and its effect is better than oral administration group and commercially available MAILUONING group.
The MAILUONING extract injection to microcirculation of mouse auricle influence (X ± SD) see the following form:
Administration group and matched group compare,
*P＜0.05,
*P＜0.01
4. the MAILUONING extract injection is to the influence of blood stasis disease hemorheology of rat
70 male rats are divided into groups 10 every group by body weight is balanced.Be respectively normal control group, blood stasis model group, MAILUONING extract injection 10,20, three dosage groups of 40g/kg, MAILUONING gastric infusion group, dosage is 20g/kg and commercially available MAILUONING ZHUSHEYE 20g/kg group.Administration every day 1 time, successive administration 7 days, normal control group and model control group are given quite volumetrical 1% poloxamer normal saline, the administration volume is 0.5mL/100g, injection speed is 2.0mL/min, water is can't help in fasting after the administration in the 6th day, chloral hydrate 350mg/kg intraperitoneal injection of anesthesia after the last administration, and the tail vein is injected 10% high molecular dextran 1.0mL/100g fast.Open breast behind the 1hr, heart extracting blood 4mL, heparin 0.1mL anticoagulant.Survey hemorheological indexes.Conclusion: the MAILUONING extract injection can significantly improve whole blood viscosity and the whole blood reduced viscosity under the different shear rates of blood stasis disease rat.
The MAILUONING extract injection to the hemorheological influence of blood stasis disease (X ± SD, n=10) see the following form:
Administration group and matched group compare,
*P＜0.05,
*P＜0.01
The MAILUONING extract injection to the hemorheological influence of blood stasis disease (X ± SD) see the following form:
Administration group and matched group compare,
*P＜0.05,
*P＜0.01
Conclusion (of pressure testing):
1, continuous 7 days intravenous administrations of MAILUONING extract injection (10,20,40g/kg) can significantly suppress the rat platelet aggregation of ADP, collagen, thrombin induction.
2, intravenous administration can significantly improve the nervous symptoms of MCAO rat to MAILUONING extract injection (10,20,40g/kg) in continuous 7 days, and its cerebral infarction is had remarkable protective effect.
3, intravenous administration can significantly suppress rat postcava thrombosis to MAILUONING extract injection (10,20,40g/kg) in continuous 7 days.
4, continuous 7 days intravenous administrations of MAILUONING extract injection (10,20,40g/kg) can high molecular dextrans cause the hemorheological indexes of rat blood stasis disease.
5, intravenous administration significantly suppressed rabbit common carotid artery thrombosis to MAILUONING extract injection (5,10,20g/kg) in continuous 7 days.
6, the mouse lung thrombosis death that collagen and adrenalin is brought out of continuous 7 days intravenous administrations of MAILUONING extract injection (15,30,60g/kg) has remarkable protective effect.
7, intravenous administration significantly improved the Mice Auricle microcirculation to MAILUONING extract injection (15,30,60g/kg) in continuous 7 days.
Continuous 7 days intravenous administrations of MAILUONING extract injection (10,20,40g/kg) can significantly suppress the rat platelet aggregation of ADP, collagen, thrombin induction; Significantly improve the nervous symptoms of MCAO rat, its cerebral infarction is had remarkable protective effect; Significantly suppress rat postcava thrombosis; Significantly improve epinephrine and cause the hemorheological indexes that rat mesentery microcirculation disturbance and high molecular dextran cause rat blood stasis disease; 5,10,20g/kg administration in continuous 7 days significantly suppresses rabbit common carotid artery thrombosis; 15,30,60g/kg administration in continuous 7 days mouse lung thrombosis death that collagen and adrenalin is brought out has remarkable protective effect, significantly improves the Mice Auricle microcirculation.In sum; the MAILUONING extract injection is in selected dosage range; significantly anticoagulant and artery and vein thrombosis have remarkable protective effect to the MCAO rat cerebral infarction, to the microcirculation disturbance effect of being significantly improved with improve every index such as blood stasis rat model whole blood viscosity.
Experimental example 1
This experimental example has illustrated the effect of MAILUONING injectable powder treatment Blood stasis of the present invention clinical trial.
One, observation index:
1) main curative effect index: (1) venation blood stasis; (2) ecchymosis; (3) hesitant pulse, pulsus deletus or heavy string, stringy and slow pulse.
2) secondary efficacy index: (1) numb limbs and tense tendons or hemiplegia; (2) tcm syndrome
3) safety indexes: (1) blood, urine, just routine, the heart, liver, renal function; (2) blood pressure.
Two, test overall design: this experimental example is clinical efficacy and the safety of investigating MAILUONING ZHUSHEYE treatment heart failure, according to " specification requirement ", will be tried routine number and be decided to be 240 examples, and 120 examples are organized in treatment, matched group 120 examples.Be set at 28 days the course of treatment.
Three, experimenter's selection and withdrawing from:
1, thromboangiitis obliterans, venous thrombosis and atherosclerotic occlusive disease Western medicine diagnose standard
(1) acra is sent out cold, is afraid of cold numbness, foot and shank distending pain and tic;
(2) malnutrition comprises that xerosis cutis, desquamation, crowfoot cracks, fine hair come off, toenail thickens, is out of shape, stops growing, muscular flaccidity or atrophy;
(3) migratory thrombophlebitis;
(4) arteriopalmus weakens or disappears;
(5) color of the leather changes;
(6) gangrene and ulcer.
2, thrombosis and sequela Western medicine diagnose standard
A, a following above nervous symptoms or sign, and continue 24 hours at least.
(1) disturbance of consciousness.
(2) vision, disturbance of visual field.
(3) paresis or hemiplegia, or diplegia (especially when brain stem damages).
(4) inclined to one side side sensory disturbance.
(5) speech disorder.
(6) dysphagia.
(7) movement disorder.
B, cerebrospinal fluid are colourless, transparent
C, the visible positive with the above auxiliary examination of the next item down change
(1) density regions of CT scan visual cue cerebral edema, cerebral ischemia pathological changes does not change and there is hemorrhagic.
(2) cerebral angiography is found one or the narrow or inaccessible change of an above trunk tremulous pulse height.
(3) brain scanning prompting cerebral infarction and except the cerebral tumor.
D, diagnosis are judged:
(1) determines diagnosis: possess A and B fully.
(2) highly possible: as to possess A to B, reach (3) item among the C.
Four, venation syndrome of blood stasis Chinese medical discrimination: be equivalent to first and second phase merging superficial thrombophlebitis person or slight gangrene of the third phase, ulcer secondary infection person.Show as twinge, localized pain, tenderness; The venation blood stasis; Ecchymosis; Purplish tongue, tongue body ecchymosis, petechia; Hesitant pulse or pulsus deletus; Squamous and dry skin; Numb limbs and tense tendons or hemiplegia; Forgetful.
Five, efficacy determination and result:
1. clinical recent recovery from illness: the clinical symptoms of blood stasis, sign disappear or basic the disappearance, are 95%.
2. produce effects: clinical symptoms, the sign of blood stasis are obviously improved, and are 70%.
3. effective: clinical symptoms, the sign of blood stasis all take a favorable turn, and are 30%.
4. invalid: clinical symptoms, the sign of blood stasis have no significant change, even increase the weight of, and are 30%.
Annotate: computing formula (nimodipine method): [integration before (integration before the treatment-treatment back integration)/treatment] * 100%.Six, conclusion: product of the present invention has clearing away heat and nourishing YIN, the effect of blood circulation promoting and blood stasis dispelling.Be used for thromboangiitis obliterans, venous thrombosis, arteriosclerosis obliterans, cerebral thrombosis and sequela etc.Pharmacodynamic test of active extract shows that product of the present invention has clearing away heat and nourishing YIN, and blood circulation promoting and blood stasis dispelling improves blood circulation and hemorheological property effect.
Experimental example 2
This experimental example explanation injectable powder long term toxicity test result of the present invention
1. dosage: according to maximal dose 100g administration in crude drug/Kg/ days.Be equivalent to 30 times of people's dosage.
2. administration time: 2 months.
3. number of animals: according to the new drug reviewing requirement, the laboratory animal number of dog is 4 or 6, selects 4 dogs for use.
4. result:
Table 1: the long poison of dog is respectively organized administration front and back serum urea nitrogen (BUN) value (mmol/L)
Normal value 1.78～8.53mmol/L of √ dog (5～25mg/dl) (1221 pages of pharmacological experimental methodologies)
Normal value 2.9～6.4mmol/L of √ people (11 pages of Chinese encyclopaedical brief introductions)
Table 2: the long poison of dog is respectively organized administration front and back serum creatinine (Cr) value (μ mol/L)
√ Cr raises and represents that it is the renal dysfunction index that Cr removes minimizing
√ dog normal value 70～180 μ mol/L
√ people's normal value 53～159 μ mol/L
5. conclusion: can find out that from 4 dogs being carried out and the long malicious experimental result of rat whole blood biochemical indicator (before and after the administration) all in normal range, illustrates animal health.
Experimental example 3
Following table 3 explanations injectable powder of the present invention and preparation method thereof and the contrast effect of existing commercially available prod on technology stability and product quality.
Product injectable powder of the present invention and commercially available product joined in glucose or the sodium chloride solution by clinical usage simultaneously compare.
Table 3
More than every the key technical indexes preparation method process stabilizing of the present invention is described, product reaches the pertinent regulations of " specification requirement of new Chinese medicine Study on Preparation ", with the product of existing method preparation in indifference qualitatively.
Experimental example 4
The stability of this experimental example explanation injectable powder of the present invention.
According to " medicine registration management way " and " specification requirement of Chinese medicine research ", take room temperature reserved sample observing method, at the clinical preliminary observation that is undertaken six months with (glass bottle) under the terms of packing by the quality standard requirement, respectively at 0,1,2 after the sample production, 3, June, each detected once, had detected altogether five times, and the result shows that injectable powder stability of the present invention is fine.Whole study on the stability is also in the middle of proceeding.The result is as follows in the preliminarily stabilised investigation; The results are shown in Table 4,5,6.
Table 4: room temperature is investigated the 1 preparation date of sample: embodiment: March 13 in 2003
Table 5: room temperature is investigated the 2 preparation dates of sample: embodiment: on March 14th, 2003
Table 6: room temperature is investigated the 3 preparation dates of sample: embodiment: on March 15th, 2003
Experimental example 5
This example is that content of total flavone is measured in the MAILUONING ZHUSHEYE
(1) method determines
Contain flavones ingredients such as luteolin in the Flos Lonicerae, Radix Scrophulariae also contains a small amount of flavones ingredient.And in preparation, can more fully be extracted according to the technology flavones ingredient.According to the literature, flavones ingredients such as luteolin have the effect that the arterial pressure of making increases and reduce venous pressure, but coronary blood flow increasing cure mainly with the function of finished product and to conform to, be the effective ingredient in the finished product, be the index of assay therefore with the total flavones.
Measure about content of total flavone, bibliographical information has: TLCS, ultraviolet spectrophotometry, colorimetry etc., and wherein in the majority with colorimetry, and mostly to be with the rutin be reference substance.Carried out the research of content of total flavone assay method in the former declaration material, it is as follows now to carry out methodological study again at the product of producing by new technology:
1. method
(1) sample preparation
Reference substance solution: precision takes by weighing 120 ℃ of control substance of Rutin 10mg that are dried to constant weight, puts in the 100ml measuring bottle, and it is an amount of to add 50% methanol, and jolting makes dissolving, and is diluted to scale, shakes up, and promptly gets (containing rutin 0.10mg among every 1ml).
Need testing solution: precision is measured this product 2ml, puts in the 25ml measuring bottle, adds 50% methanol and is diluted to scale, shakes up, promptly.
The negative control solution that lacks Flos Lonicerae: take by weighing Radix Achyranthis Bidentatae, Herba Dendrobii, each 100g of Radix Scrophulariae,, make the MAILUONING ZHUSHEYE negative sample that lacks Flos Lonicerae, make the negative control solution that lacks Flos Lonicerae according to the preparation method of aforementioned need testing solution according to embodiment 1-1 method.
The jack to jack adapter contrast solution that lacks Flos Lonicerae, Radix Scrophulariae: take by weighing Radix Achyranthis Bidentatae, each 100g of Herba Dendrobii, make the MAILUONING ZHUSHEYE that lacks Flos Lonicerae, Radix Scrophulariae, make the jack to jack adapter contrast solution that lacks Flos Lonicerae, Radix Scrophulariae according to the preparation method of aforementioned need testing solution according to technology
(2) measure
The preparation of standard curve: precision is measured above-mentioned reference substance solution 1.0,2.0,3.0,4.0,5.0ml, put respectively in the 10ml measuring bottle, the sodium nitrite solution 0.3ml of accurate adding 5% shakes up, placed 6 minutes, add 10% aluminum nitrate solution 0.6ml, shake up, placed 6 minutes, add 8.6% sodium hydroxide solution 3ml, adding 50% methanol and be diluted to scale, shake up, is blank with the reagent corresponding.According to spectrophotography (appendix VB of Chinese Pharmacopoeia version in 2000), measure trap at the wavelength place of 510nm, be vertical coordinate with the trap, concentration is abscissa, the drawing standard curve.
The mensuration of need testing solution: precision is measured need testing solution 3ml, puts in the 10ml measuring bottle, adds 50% methanol and is diluted to scale, shakes up, as blank; Precision is measured need testing solution 3ml in addition, puts in the 10ml measuring bottle, and the method under the sighting target directrix curve preparation from " adding 50% methanol to 5ml ", is measured trap in accordance with the law, reads the weight that is equivalent to rutin the need testing solution from standard curve, calculates, promptly.
(2) the content of total flavone assay method is learned and is investigated
1. reagent and instrument
Reagent: aluminum nitrate: analytical pure, Tianjin chemical reagent three factories, lot number: 20011127;
Sodium nitrite: analytical pure, Tianjin Chemical Reagents Factory No.1, lot number: 900815
Sodium hydroxide: analytical pure, Beijing Chemical Plant, lot number: 931016
Control substance of Rutin: Nat'l Pharmaceutical ﹠ Biological Products Control Institute provides, 0080-9705
Instrument: day island proper Tianjin UV-2210 ultraviolet-visible spectrophotometer
2. the selection of color condition
In the methodological study originally to 1. 5% sodium nitrite solution; 2. 10% aluminum nitrate solution; 3. the consumption of three kinds of test solutions of sodium hydroxide test solution of 8.6% adopts L
(9)(3
4) orthogonal experiment carried out preferably the addition of three kinds of test solutions, color developing effect was ideal when proof added 5% sodium nitrite solution 0.3ml, 10% aluminum nitrate solution 0.6ml, 8.6% sodium hydroxide test solution 3ml respectively, so still adopt this condition to develop the color.
3 color stabilities are investigated
(lot number: 030212) 2ml, the coloration method operation of shining the front was sentenced the time sweep mode at 510nm at once later at adding sodium hydroxide test solution 6ml and measured trap in 0～25 minute to get need testing solution.Measurement result shows, trap is maximum and more stable in back 15 minutes of colour developing, and trap begins to descend afterwards.Consistent with the result of study in the former declaration material.With reference to " list of references of the relevant determination of total flavonoids of the content assaying method in PAISHI KELI quality standard of Chinese pharmacopoeia version in 2000 and other is measured trap after being chosen in colour developing immediately
4 measure the selection of wavelength
The accurate control substance of Rutin solution 3ml that draws 0.1141mg/ml, finished product 030315 batch sample solution, according to aforementioned coloration method operation, wave-length coverage interscan at 400～600nm, record control substance of Rutin solution and absorption maximum is arranged at the 506nm place, peak shape is more flat, is a steamed bread peak, and trap changes less than 1.3% in ± 5nm wave-length coverage; Sample solution colour developing back absorption maximum is at 515nm, and absworption peak is similarly a steamed bread peak, and trap changes less than 1.0% in the ± 5nm wave-length coverage.According to another bibliographical information, the colorimetry assay more options of total flavones are measured trap at the 510nm place, also select 510nm to measure wavelength so this product content of total flavone is measured.
The preparation of 5 standard curves
Precision takes by weighing in 120 ℃ of control substance of Rutin 11.41mg that are dried to constant weight in the 100ml measuring bottle, adds 50% dissolve with methanol and is diluted to scale (0.1141mg/ml).Accurate absorption 1.0,2.0,3.0,4.0,5.0ml are in the 10ml measuring bottle, the sodium nitrite solution 0.3ml of accurate adding 5%, shake up, placed 6 minutes, the aluminum nitrate solution 0.6ml of accurate adding 10%, shake up, placed 6 minutes, and added 8.6% sodium hydroxide solution 3ml, add 50% methanol and be diluted to scale, shaking up, is blank with the reagent corresponding.According to spectrophotography (appendix VB of Chinese Pharmacopoeia version in 2000), measure trap at the wavelength place of 510nm, be vertical coordinate with trap (A), concentration (B) is abscissa, the drawing standard curve.The results are shown in following table:
| The concentration of control substance of Rutin (mg/l) | Trap (Abs) |
| 0.01141 | 0.1662 |
| 0.02282 | 0.2989 |
| 0.03423 | 0.4366 |
| 0.04564 | 0.5788 |
| 0.05705 | 0.7118 |
Calculate to such an extent that standard curve is: A=12.006B+0.028, r=0.9999 by the said determination result.
See Fig. 1 for details.
6 stability tests
(lot number: 030314) 2ml puts in the 25ml measuring bottle accurate absorption sample solution, and the methanol of adding 50% shakes up to scale.Draw 2ml 0,4,8,22,25,30 hour precision respectively, develop the color according to preceding method, need testing solution (2ml → 10ml with respective concentration, 50% methanol solution is diluted to scale) be blank, measure trap at 510nm wavelength place, the substitution standard curve calculates, and measurement result sees the following form:
| Time (hour) | Trap (Abs) | General flavone content (mg/ml) |
| 0 | 0.327 | 1.557 |
| 4 | 0.324 | 1.547 |
| 8 | 0.323 | 1.536 |
| 22 | 0.323 | 1.536 |
| 25 | 0.321 | 1.525 |
| 30 | 0.325 | 1.546 |
Conclusion: the meansigma methods of the general flavone content of six time points is 1.541mg/ml, RSD=0.72%.The result shows that need testing solution content of total flavone in 30 hours is stable.
The test of 7 precision
(lot number: 030313) 2ml puts in the 25ml measuring bottle accurate absorption sample solution, and the methanol of adding 50% shakes up to scale.Accurate totally 5 parts of the 3ml that draw according to the preceding method colour developing, measure trap at the wavelength place of 510nm, and the substitution standard curve calculates, and measurement result sees the following form:
| Numbering | Trap (Abs) | General flavone content (mg/ml) |
| 1 | 0.306 | 1.447 |
| 2 | 0.326 | 1.551 |
| 3 | 0.314 | 1.489 |
| 4 | 0.322 | 1.530 |
| 5 | 0.323 | 1.536 |
| 6 | 0.319 | 1.515 |
Conclusion: the meansigma methods of 6 mensuration is 1.511mg/ml, RSD=2.51%.The result shows that this method precision is good.
8 replica tests
Get embodiment 2-1 to 2-6 product solution 2ml, totally 6 parts, put respectively in the 25ml measuring bottle, add 50% methanol to scale, shake up.Precision is measured 2ml in the 10ml measuring bottle, adds 50% methanol to 5ml, adds 5% sodium nitrite solution 0.3ml, shake up, placed 6 minutes, the aluminum nitrate solution 0.6ml of accurate adding 10%, shake up, placed 6 minutes, add 8.6% sodium hydroxide solution 3ml, add 50% methanol and be diluted to scale, shake up, according to spectrophotography (appendix VB of Chinese Pharmacopoeia version in 2000), wavelength place at 510nm measures trap, the substitution standard curve calculates, and measurement result sees the following form:
| Trap (Abs) | General flavone content (mg/ml) | |
| Embodiment 2-1 | 0.315 | 1.494 |
| Embodiment 2-2 | 0.322 | 1.530 |
| Embodiment 2-3 | 0.326 | 1.551 |
| Embodiment 2-4 | 0.318 | 1.510 |
| Embodiment 2-5 | 0.319 | 1.515 |
| Embodiment 2-6 | 0.322 | 1.530 |
Conclusion: the meansigma methods of 6 replications of general flavone content of this batch sample is 1.522mg/ml, RSD=1.30%.The result shows that the repeatability of this method is good.
9 recovery tests
The preparation of control substance of Rutin solution: precision takes by weighing through 120 ℃ of control substance of Rutin 17.35mg that are dried to constant weight in the 25ml measuring bottle, adds dissolve with methanol and is diluted to scale, shakes up, and promptly gets (0.694mg/ml).
030312) 1.0,1.5, each 2 parts of 2.0ml the preparation of need testing solution: precision is measured sample solution (lot number:, put in the 25ml measuring bottle, each accurate above-mentioned control substance of Rutin solution 2.0,2.0,3.0,3.0,3.5,3.5ml of adding respectively adds 50% methanol and is diluted to scale, shake up, promptly.
Measure: precision is measured above-mentioned need testing solution 2ml, according to aforementioned method suggested colour developing, measures trap at the wavelength place of 510nm, and the substitution standard curve with the following formula calculate recovery rate, the results are shown in table 20, and the result shows that this method response rate is good.
Table 20. total flavones determination of recovery rates result
Content of total flavone is measured in 10 samples
Measure content of total flavone among the embodiment 2-1 to 2-5, the results are shown in following table:
The determination of total flavonoids result:
| General flavone content meansigma methods (mg/ml) | |
| Embodiment 2-1 | 3.12 |
| Embodiment 2-2 | 2.52 |
| Embodiment 2-3 | 3.06 |
| Embodiment 2-4 | 2.92 |
| Embodiment 2-5 | 2.41 |
Five batch sample general flavone content meansigma methodss are 2.81mg/ml.
Experimental example 6
This example is the assay of Flos Lonicerae in the MAILUONING ZHUSHEYE
Adopting the HPLC method, is index with the chlorogenic acid, measures the content of Flos Lonicerae.Now chlorogenic acid contents in five batches of products of embodiment 1-1 to embodiment 1-4, embodiment 2-1 is measured.
1, instrument and reagent
Waters2 695 high performance liquid chromatographs; Waters 2996 diode array detector;
The Empower data processing software;
Methanol (chromatographically pure): Fisher Scientific company, lot number: 020215
Water: double distilled water;
Glacial acetic acid (AR level), Suzhou City's second chemical institute, lot number: 000201
The chlorogenic acid reference substance: Nat'l Pharmaceutical ﹠ Biological Products Control Institute provides, for assay usefulness, lot number: 0753-200111.
2, chromatographic condition
Chromatographic column: Kromasil C18 (Φ 4.6 * 250mm, 5 μ m)
Column temperature: 25 ℃
Mobile phase: methanol-water-glacial acetic acid (15:85:1), flow velocity: 0.8ml/min
Sample size: 10 μ l
Wavelength: 330nm
3, sample preparation
Reference substance solution: it is an amount of to get the chlorogenic acid reference substance, adds methanol and makes the solution that contains 50 μ g among every 1ml.
Need testing solution: precision is measured sample solution 5ml, puts in the 25ml measuring bottle, adds mobile phase and is diluted to scale, shakes up, promptly.
The negative control solution that lacks Flos Lonicerae: take by weighing Radix Achyranthis Bidentatae, Herba Dendrobii, each 10g of Radix Scrophulariae, make the MAILUONING ZHUSHEYE that lacks Flos Lonicerae, make the negative control solution that lacks Flos Lonicerae according to the preparation method of aforementioned need testing solution according to technology.
4, negative control test
Under aforementioned chromatographic condition, accurate each 10 μ l of negative control solution that draw above-mentioned reference substance solution and scarce Flos Lonicerae, inject chromatograph of liquid, record HPLC figure, the result shows that (about 16.6 minutes) have a little and wide chromatographic peak to negative sample at retention time place, chlorogenic acid peak, but is difficult to its peak area integration is come out because of peak area is too little.According to " study of tcm new drug guide ", blank assay allows therefore can think that negative sample is noiseless substantially to the mensuration of chlorogenic acid in the sample below 5% to the interference of sample, and is consistent with former result of study.
5, sample determination and result
By preceding method five batch samples are measured, measurement result sees the following form:
| Chlorogenic acid contents meansigma methods (μ g/ml) | |
| Embodiment 1-1 | 181.5 |
| Embodiment 1-2 | 183.5 |
| Embodiment 1-3 | 203.6 |
| Embodiment 1-4 | 157.2 |
| Embodiment 2-1 | 143.3 |
| Meansigma methods | 173.8 |
According to the sample that Chinese patent CN92107848.X makes, the chlorogenic acid content meansigma methods is 102.2 μ g/ml, the product that makes well below the inventive method.
Experimental example 7
This example is the assay of Radix Scrophulariae in the MAILUONING ZHUSHEYE
1. instrument and reagent
Waters 2695 high performance liquid chromatographs; Waters 2996 diode array detector;
The Empower data processing software;
Reagent:
Methanol (chromatographically pure): Fisher Scientific company, lot number: 020215
Water: double distilled water;
Glacial acetic acid (AR level), Suzhou City's second chemical institute, lot number: 000201
The cinnamic acid reference substance: Nat'l Pharmaceutical ﹠ Biological Products Control Institute provides, for assay usefulness, lot number: 0786-9802.
2. chromatographic condition
Chromatographic column: Kromasil C18 (Φ 4.6 * 250mm, 5 μ m)
Column temperature: 25 ℃
Mobile phase: methanol-water-glacial acetic acid (45:55:0.2), flow velocity: 0.8ml/min
Sample size: 10 μ l
Wavelength: 273nm
3. sample preparation
Reference substance solution: it is an amount of to get the cinnamic acid reference substance, adds methanol and makes the solution that contains 50 μ g among every 1ml.
Need testing solution: sample thief stock solution direct injected.
The negative control solution that lacks Radix Scrophulariae: take by weighing Radix Achyranthis Bidentatae, Herba Dendrobii, each 10g of Flos Lonicerae, make the MAILUONING ZHUSHEYE that lacks Radix Scrophulariae, promptly get the negative control solution that lacks Radix Scrophulariae according to technology.
4. negative control test
Under aforementioned chromatographic condition, accurate each 10ul of negative control solution that draws above-mentioned reference substance solution and scarce Radix Scrophulariae, inject the HPLC instrument, record HPLC figure, the result shows that (about 18.6 minutes) no chromatographic peak occurs negative sample at retention time place, cinnamic acid peak, illustrates that negative sample is noiseless to the mensuration of cinnamic acid in the sample.
5. sample determination and result
| The content meansigma methods of cinnamic acid (μ g/ml) | |
| Embodiment 2-2 | 40.5 |
| Embodiment 2-3 | 41.8 |
| Embodiment 2-4 | 42.8 |
| Embodiment 2-5 | 46.2 |
| Embodiment 2-6 | 50.8 |
Three crowdes of sample determination results that make according to Chinese patent CN92107848.X:
| Cinnamic acid content meansigma methods (μ g/ml) | |
| 1 | 38.6 |
| 2 | 36.0 |
| 3 | 42.0 |
6. conclusion:
Negative sample is noiseless to the mensuration of cinnamic acid.The sample cinnamic acid content meansigma methods that five batches of new technologies of this product are produced is 44.4 μ g/ml, and the cinnamic acid content meansigma methods that is higher than Chinese patent CN92107848.X explained hereafter product is 39.1 μ g/ml.
Experimental example 8
This example is the assay of Radix Achyranthis Bidentatae in the MAILUONING ZHUSHEYE
1. instrument and reagent
Waters 2695 high performance liquid chromatographs; Waters 2996 diode array detector;
The Empower data processing software;
Reagent:
Acetonitrile (chromatographically pure): Fisher Scientific company, lot number: 020109.
Water: double distilled water;
Ecdysterone reference substance: provide by natural pharmacology system of College of Pharmacy, Beijing Univ.
2. chromatographic condition
Chromatographic column: Kromasil C18 (Φ 4.6 * 250mm, 5 μ m),
Column temperature: 30 ℃,
Mobile phase: acetonitrile-water (1:5.4), flow velocity: 1.0ml/min,
Sample size: 10 μ l
Wavelength: 243nm
3. sample preparation
Reference substance solution: it is an amount of to get the ecdysterone reference substance, adds methanol and makes the solution that contains 50 μ g among every 1ml.
Need testing solution: precision is measured sample solution 25ml, put in the separatory funnel, add sodium chloride 2.5g, jolting makes dissolving, with water saturated n-butanol extraction six times, each 25ml merges n-butyl alcohol liquid, with the saturated water 20ml washing of n-butyl alcohol, cleaning mixture extracts with water saturated n-butyl alcohol 20ml, merges n-butyl alcohol liquid, puts evaporate to dryness in the water-bath, residue adds 60% methanol makes dissolving, be transferred in the 25ml measuring bottle, add 60% methanol, shake up to scale, filter with 0.45 μ m microporous filter membrane, filtrate is as need testing solution.
The negative control solution that lacks Radix Achyranthis Bidentatae: take by weighing Radix Scrophulariae, Herba Dendrobii, each 50g of Flos Lonicerae, make the MAILUONING ZHUSHEYE that lacks Radix Achyranthis Bidentatae, make the negative control solution of scarce Radix Achyranthis Bidentatae according to the preparation method of need testing solution according to technology.
4. negative control test
Under aforementioned chromatographic condition, the accurate negative control solution 10 μ l that draw above-mentioned scarce Radix Achyranthis Bidentatae, inject chromatograph of liquid, record HPLC chromatogram, the result shows that (about 30.3 minutes) no chromatographic peak occurs negative sample at retention time place, ecdysterone peak, illustrates that negative sample is noiseless to the mensuration of ecdysterone in the sample.
5. sample determination and result
By preceding method five batch samples of the present invention are measured, measurement result sees the following form
| The content meansigma methods of ecdysterone (μ g/ml) | |
| Embodiment 3-1 | 48.7 |
| Embodiment 3-2 | 47.0 |
| Embodiment 3-3 | 48.0 |
| Embodiment 3-4 | 48.2 |
| Embodiment 3-5 | 46.8 |
| Meansigma methods | 47.7 |
The sample determination that makes according to Chinese patent CN92107848.X the results are shown in following table:
| Ecdysterone content meansigma methods (μ g/ml) | |
| 1 | 25.4 |
| 2 | 10.0 |
| 3 | 30.2 |
| 4 | 21.6 |
| Meansigma methods | 21.8 |
6. conclusion: the sample ecdysterone content meansigma methods of explained hereafter of the present invention is 47.7 μ g/ml, is higher than the ecdysterone content that Chinese patent CN92107848.X makes sample far away.
Experimental example 9
This experimental example is given white mice LD for product acute toxicity test MAILUONING extract injection of the present invention intravenous injection
50Greater than 1600g/kg, according to the weight, 480 times of the consumption that is equivalent to be grown up; Lumbar injection is given white mice LD
50Greater than 2000g/kg, according to the weight, 606 times of the consumption that is equivalent to be grown up.
Test objective:
Observation was tried thing and given toxic reaction and the death condition that (intravenous injection and lumbar injection) produced behind the white mice maximum dosage-feeding in one day, to examine or check its clinical application safety.
Test material:
1. tried thing
Title: MAILUONING extract injection
Specification and content: contain extract 17g among the 100mL, be equivalent to crude drug in whole 2000g.
Lot number: 030211
The unit of providing: Harbin work of nature academy of science
2. animal subject
Title: white mice
Strain: Kunming kind
Sex: male and female dual-purpose
Body weight: 18-22g
Every treated animal number: 20
The quality certification number: SCXK (the Liao Dynasty) 2003-008
The unit of providing: Shenyang Pharmaceutical University's Experimental Animal Center
Test method and result:
1, intravenous administration: 40 mices are divided into 2 groups by body weight, sex equilibrium, 20 every group, male and female half and half, administration group mouse mainline MAILUONING extract injection, the administration volume is 0.4ml/10g, and injection speed is 0.06mL/s, and dosage is 800g/kg; Matched group is given isometric 1% poloxamer, 0.9% sodium chloride injection, distinguishes administration again 1 time after 8 hours.One week of observation after the administration, find to darken (near the medicine color) except that mice activity minimizing, palpitating speed, urine, outside the recovery normally, surplus discovery is tried mice obvious toxic reaction after 20 minutes, and 20 mices all survive.Control group mice body weight and administration group mice body weight there was no significant difference after seven days do not find under the postmortem naked eyes that being tried the mice main organs has obvious pathological changes.The mice body weight change is as shown in the table.
MAILUONING extract injection intravenous administration to the influence of white mice body weight (X ± SD) see the following form:
2, intraperitoneal injection: mice group is the same.Administration group mouse peritoneal injection MAILUONING extract injection, the administration volume is 0.5ml/10g, dosage is 1000g/kg; Matched group is given isometric 0.9% sodium chloride injection, distinguishes administration again 1 time after 8 hours.Observe a week after the administration, toxic reaction is the same.20 mices all survive, and control group mice body weight and administration group mice body weight there was no significant difference after seven days do not find under the postmortem naked eyes that being tried the mice main organs has obvious pathological changes.The mice body weight change is as shown in the table.
MAILUONING extract injection intraperitoneal injection to the influence of white mice body weight (X ± SD) see the following form:
Conclusion:
White mice LD is given in the intravenous injection of MAILUONING extract injection
50Greater than 1600g/kg, consumption 480 times according to the weight is equivalent to be grown up; Lumbar injection is given white mice LD
50Greater than 2000g/kg, according to the weight, 606 times of the consumption that is equivalent to be grown up.
Claims (9)
1, the Chinese medicine of a kind of treatment or prevention thrombotic disease is characterized in that, described Chinese medicine is that the raw material components with following weight portion extracts and obtains:
Radix Achyranthis Bidentatae 100-1000 Radix Scrophulariae 100-1000
Herba Dendrobii 100-1000 Flos Lonicerae 100-1000
The extraction of effective ingredient realizes as follows:
1) Radix Achyranthis Bidentatae in the above-mentioned raw materials component, Radix Scrophulariae, Herba Dendrobii are rinsed well, cut into slices or section or powder, drop in the extraction pot in the lump with Flos Lonicerae, adding the 0%-99.5% ethanol water that 1-10 doubly measures soaked after 1 hour, 40-85 ℃ of following heating and refluxing extraction 1-5 time, the each extraction 1-2 hour filters filtrate for later use; Merging filtrate, being concentrated into relative density is 1.10-1.20 in the time of 80 ℃;
2) concentration of adding 1/10 volume is the 0.5%-10% aqueous gelatin solution in concentrated solution, stirs, and places 12-24 hour for 0-10 ℃, and is centrifugal, gets supernatant;
3) add 95% ethanol, stir, make the supernatant ethanol content at 75-85%, 0-10 ℃ left standstill 12-24 hour, the leaching supernatant, and reclaiming ethanol and being concentrated into relative density is 1.10-1.20 in the time of 80 ℃;
4) ethyl acetate of 1-3 times of concentrated solution volume of adding extracts 3-7 time, extracts 5-15 minute at every turn, merges supernatant, reclaims ethyl acetate, and removes ethyl acetate, is concentrated into thick paste in the time of 80 ℃, is effective ingredient.
2, Chinese medicine according to claim 1 is characterized in that, cinnamic acid content is 40-51 μ g/mL in the described Chinese medicine, and chlorogenic acid content is 140-205 μ g/mL.
3, Chinese medicine according to claim 2 is characterized in that, sterone content is 45-50 μ g/mL in the described Chinese medicine.
4, Chinese medicine according to claim 3 is characterized in that, in the described Chinese medicine, and content of beary metal≤10ppm, arsenic salt≤1ppm, residue on ignition≤0.8%g/mL, total solid 〉=16.3mg/mL.
5, Chinese medicine according to claim 4 is characterized in that, the protein of described Chinese medicine, tannin, resin, oxalates, potassium ion meet the pertinent regulations under 2000 editions injection items of Chinese Pharmacopoeia.
6, a kind of preparation method of Chinese medicine is characterized in that, the extraction of effective ingredient realizes as follows:
1) with the raw material components of following weight portion
Radix Achyranthis Bidentatae 100-1000 Radix Scrophulariae 100-1000
Herba Dendrobii 100-1000 Flos Lonicerae 100-1000
Rinse well, cut into slices or section or powder, drop in the lump in the extraction pot with Flos Lonicerae, add the 0%-99.5% ethanol water that 1-10 doubly measures and soak after 1 hour, 40-85 ℃ of following heating and refluxing extraction 1-5 time extracted filtration, filtrate for later use 1-2 hour at every turn; Merging filtrate, being concentrated into relative density is 1.10-1.20 in the time of 80 ℃;
2) concentration of adding 1/10 volume is the 0.5%-10% aqueous gelatin solution in concentrated solution, stirs, and places 12-24 hour for 0-10 ℃, and is centrifugal, gets supernatant;
3) add 95% ethanol, stir, make the supernatant ethanol content at 75-85%, 0-10 ℃ left standstill 12-24 hour, the leaching supernatant, and reclaiming ethanol and being concentrated into relative density is 1.10-1.20 in the time of 80 ℃;
4) ethyl acetate of 1-3 times of concentrated solution volume of adding extracts 3-7 time, extracts 5-15 minute at every turn, merges supernatant, reclaims ethyl acetate, and removes ethyl acetate, is concentrated into thick paste in the time of 80 ℃, is effective ingredient.
7, a kind of preparation method of Chinese medicine is characterized in that, the method for using macroporous resin to carry out purification replaces the step 3) described in the claim 6.
8, a kind of preparation method of Chinese medicine is characterized in that, the leaching process of described effective ingredient is:
1) with raw material components: Radix Achyranthis Bidentatae, Radix Scrophulariae, Herba Dendrobii among Radix Achyranthis Bidentatae 100-1000, Radix Scrophulariae 100-1000, Herba Dendrobii 100-1000 and the Flos Lonicerae 100-1000 are rinsed well, cut into slices or section or powder, drop in the extraction pot in the lump with Flos Lonicerae, adding the 0%-99.5% ethanol water that 1-10 doubly measures soaked after 1 hour, 40-85 ℃ of following heating and refluxing extraction 1-5 time, the each extraction 1-2 hour filters filtrate for later use; Merging filtrate, being concentrated into relative density is 1.10-1.20 in the time of 80 ℃;
2) concentration of adding 1/10 volume is the 0.5%-10% aqueous gelatin solution in concentrated solution, stirs, and places 12-24 hour for 0-10 ℃, and is centrifugal, gets supernatant;
3) supernatant is carried out purification by macroporous resin, obtain effective ingredient, be equipped with adjuvant and prepare injectable powder or injection.
9, a kind of preparation method of Chinese medicine is characterized in that, the leaching process of described effective ingredient is:
1) with raw material components: Radix Achyranthis Bidentatae, Radix Scrophulariae, Herba Dendrobii among Radix Achyranthis Bidentatae 100-1000, Radix Scrophulariae 100-1000, Herba Dendrobii 100-1000 and the Flos Lonicerae 100-1000 are rinsed well, cut into slices or section or powder, drop in the extraction pot in the lump with Flos Lonicerae, add water logging bubble that 1-10 doubly measures after 1 hour, 40-85 ℃ of following heating and refluxing extraction 1-5 time, the each extraction 1-2 hour filters filtrate for later use; Merging filtrate, being concentrated into relative density is 1.10-1.20 in the time of 80 ℃;
2) in concentrated solution, add ZTC pretreating agent A component, boil 15-20 minute after, left standstill 4-5 hour, centrifugalize gets the supernatant; Add ZTC pretreating agent A component again, boil 15-20 minute after, left standstill 3 hours, filter, add ZTC clarifier B component in the filtrate, be heated to and boil, placed 4-6 hour, centrifugal after-filtration, filtrate are behind multilamellar filter paper filter press, and being concentrated into proportion is 1.10-1.20 in the time of 80 ℃;
3) add 95% ethanol, stir, make the supernatant ethanol content at 75-85%, 0-10 ℃ left standstill 12-24 hour, the leaching supernatant, and reclaiming ethanol and being concentrated into relative density is 1.10-1.20 in the time of 80 ℃;
4) carry out purification by macroporous resin, obtain effective ingredient, be equipped with adjuvant and prepare injectable powder or injection.
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| RP-HPLC法测定脉络宁注射液中滨蒿内酯的含量. 李文莉，汪文涛，雷玉萍，蒋 丽.中草药，第35卷第7期. 2004 |
| RP-HPLC法测定脉络宁注射液中滨蒿内酯的含量. 李文莉，汪文涛，雷玉萍，蒋 丽.中草药，第35卷第7期. 2004 * |
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