CN100544741C - Herba Ixeritis Sonchifoliae total flavones and preparation method thereof and the freeze-dried powder and the detection method that contain it - Google Patents
Herba Ixeritis Sonchifoliae total flavones and preparation method thereof and the freeze-dried powder and the detection method that contain it Download PDFInfo
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Abstract
The invention discloses a kind of Herba Ixeritis Sonchifoliae total flavones and preparation method thereof, this Herba Ixeritis Sonchifoliae total flavones contains luteolin-7-O-beta d glucopyranosiduronic acid glycosides (C
21H
18O
12) be no less than 8.00%.The invention also discloses the injection freeze-dried powder that contains this Herba Ixeritis Sonchifoliae total flavones, component is Herba Ixeritis Sonchifoliae total flavones 20g, and mannitol 60g adds the injection water to 2000ml, and every contains refining Herba Ixeritis Sonchifoliae total flavones 20mg.The present invention also discloses the preparation method and the method for quality control of this injection freeze-dried powder.
Description
Technical field
The present invention relates to a kind of Herba Ixeritis Sonchifoliae total flavones and preparation method thereof, the invention still further relates to the Ixers sonchifolia freeze-dried powder that contains this Herba Ixeritis Sonchifoliae total flavones, belong to pharmaceutical field.The present invention also relates to the preparation method and the method for quality control of this freeze-dried powder.
Background technology
Herba Ixeritis Sonchifoliae is this (Ixeris sonchifolia (Bge) Hance) dry herb of 1-2 SHENGCAO, has heat-clearing and toxic substances removing, and the effect of reducing swelling and alleviating pain mainly contains compositions such as flavone, adenosine, organic acid, coumarin, aminoacid, plant sterol, saccharide.Modern medicine study shows that flavone compound has the expansion heart, cerebrovascular in the Herba Ixeritis Sonchifoliae, increases coronary flow, and anticoagulant suppresses effects such as thrombus in vivo formation.Existing market has the medicine DIEMAILING ZHUSHEYE, but because active component is indeterminate, so be difficult to its quality of control.The inventor isolates 2 kinds of neoflavone glycoside compounds (luteolin 7-O-β-D-pyranglucoside, luteolin 7-O-beta d glucopyranosiduronic acid glycosides) from refining Herba Ixeritis Sonchifoliae total flavones, experimental results show that through pharmacologically active two kinds of neoflavone glycosides have obvious protective effect to Acute Myocardial Ischemia in Rats.In view of the above, can determine effective site, improve the content of effective site, thereby make Ixers sonchifolia freeze-dried powder, make its requirement that reaches Chinese medicine, guarantee safe and effectively, curative effect is better than DIEMAILING ZHUSHEYE.
Summary of the invention
The purpose of this invention is to provide a kind of Herba Ixeritis Sonchifoliae total flavones.
Another object of the present invention provides the preparation method of this Herba Ixeritis Sonchifoliae total flavones.
Another purpose of the present invention provides a kind of freeze-dried powder that contains this Herba Ixeritis Sonchifoliae total flavones.
Another object of the present invention provides the preparation method of this Ixers sonchifolia freeze-dried powder.
Another purpose of the present invention provides the method for quality control of this Ixers sonchifolia freeze-dried powder.
Herba Ixeritis Sonchifoliae total flavones of the present invention is prepared by following method: get the Herba Ixeritis Sonchifoliae medical material and decoct with water, with decocting liquid, be concentrated into every 1ml and be equivalent to crude drug in whole 1g, put coldly, stir down and add ethanol, make to contain alcohol and measure and reach 80%, leave standstill, filter, add alkali under filtrate is stirred and regulate pH value to 11, leave standstill, centrifugal, precipitate adds 10 times of water gagings, stir, centrifugal, precipitate adds ethanol to be made and contains the alcohol amount and reach 90%, add acid under stirring, regulate pH value to 5, centrifugal, centrifugal liquid adds aqueous slkali under stirring, regulate pH value to 6.5, filter, decompression filtrate recycling ethanol is to there not being the alcohol flavor, be dissolved in water, freezing, room temperature is melted, and decompression filters, the filtrate lyophilization, promptly get Herba Ixeritis Sonchifoliae total flavones, this product is fallow amorphous powder or porous solid shape thing, wherein contains luteolin-7-O-beta d glucopyranosiduronic acid glycosides (C
21H
18O
12) be no less than 8.00%.
Preferably prepare: get the Herba Ixeritis Sonchifoliae medical material, decoct with water secondary, add 12 times of water gagings for the first time, decocted 1 hour, add 10 times of water gagings for the second time, decocted 1 hour by following method; Collecting decoction is concentrated into every 1ml and is equivalent to crude drug in whole 1g, puts cold, stir down and add ethanol, make to contain alcohol and measure and reach 80%, left standstill 12 hours, decompression filters, and filtrate adds 10% calcium oxide breast under stirring, and regulates pH value to 11.0, leave standstill 0.5 hour, centrifugal, precipitate adds 10 times of water gagings, stirred 10 minutes, centrifugal, precipitate adds ethanol to be made and contains alcohol amount and reach 90%, adds 50% sulfuric acid solution under stirring, regulate pH value to 5.0, centrifugal, centrifugal liquid adds 40% sodium hydroxide solution under stirring, and regulates pH value to 6.5, filter, decompression filtrate recycling ethanol adds the injection water and is dissolved to 100ml, freezing 24 hours to there not being the alcohol flavor, room temperature is melted, decompression filters, and the filtrate lyophilization gets Herba Ixeritis Sonchifoliae total flavones.
The injection freeze-dried powder that contains above-mentioned Herba Ixeritis Sonchifoliae total flavones of the present invention contains Herba Ixeritis Sonchifoliae total flavones and is no less than 80.0%.
The preparation method of this Ixers sonchifolia freeze-dried powder for injection comprises the following steps:
Get Herba Ixeritis Sonchifoliae total flavones 20g, mannitol 60g, add the injection water and make dissolving in right amount, 100 ℃ of steam sterilizations 30 minutes add the injection water and are settled to 2000ml, and decompression filters, and filtrate is aseptic subpackaged in cillin bottle, lyophilization, and close plug, gland is made 1000.
The foundation of its dosage form selection is as follows with the trial test result:
(1) based on the definite curative effect of domestic existing medicine " DIEMAILING ZHUSHEYE " clinical application for many years, now change aqueous injection into lyophilized injectable powder, improved product quality and stability thereof, guarantee safe and reliablely, help storing transportation.
(2) to the safety testing of injection Herba Ixeritis Sonchifoliae intravenously administrable, trial tests such as acute safety, undue toxicity, allergy and haemolysis have been carried out in safety test with reference to " Chinese medicine development guideline (trying) " promulgated by the ministries or commissions of the Central Government, the result shows, every safety testing behind the injection Herba Ixeritis Sonchifoliae intravenously administrable is all negative, guarantees safe and reliable.
Comprehensive The above results, the Herba Ixeritis Sonchifoliae medical material is prepared injection Herba Ixeritis Sonchifoliae behind extraction, separation, purification, and intravenously administrable possesses its timeliness and curative effect advantage.Long term storage can be guaranteed stability of formulation, and demonstrates safe and reliable.So said preparation is selected freeze-dried powder, the intravenous route administration is optimal dosage form.
(1) Study on extraction process
Contain compositions such as flavonoid, adenosine, organic acid in the Herba Ixeritis Sonchifoliae medical material, wherein with flavone compound as effective ingredient.In the solvents such as flavone compound is generally soluble in water, methanol, Diluted Alcohol.Therefore, extraction solvent is defined as water.Investigated the influence of amount of water, extraction time, extraction time in the technical study to yield, determine that through orthogonal test optimum condition is to extract 2 times, each 1 hour, add for the first time 12 times of amounts of water, add for the second time 10 times of amounts of water, this condition can guarantee that 80% above flavones ingredient is extracted out, saves time the energy and cost.
(2) research of separating technology
Except that flavones ingredient, also contain water-soluble components such as polysaccharide, protein, tannin in the extracting solution, we have selected two kinds of separation methods for use in the experiment, have carried out the contrast experiment.The result shows that the clarifier impurities removing method is more loaded down with trivial details, and the polysaccharide composition is not had adsorption, and the removal of impurity is incomplete, and commercial production does not adopt.Select the pure sedimentation method removal of impurity for use, easy and simple to handle, use repeatedly after ethanol is recyclable, reduce cost, and removal of impurity effect is better than clarifier.For this reason, this technology has been selected the deimpurity method of ethanol precipitation for use, and medicinal liquid is concentrated into every 1ml and contains the 1g medical material, and adding ethanol is 80% the best to containing the alcohol amount.
(3) research of purifying process
As effective ingredient,, select for use macroporous adsorbent resin method and calcium oxide breast method to carry out the purification contrast experiment flavone compound in the preparation according to the experience of separating and purifying flavone constituents.The result shows that the Amberlyst process operation is easier, be applicable to from the aqueous solution of large volume and collect flavone compound, but its general flavone content of purified sample only is about 50%.And its general flavone content of sample of alkali acid system purification gained can reach about 75%, compares, and the method purification that adopts macroporous resin aborning not only will be to used resin repeated regeneration or renewal, and yield is low.And alkali acid system gained sample purity is easy to reach the injection requirement, in order to improve the purity of sample, to calcium oxide breast adjust pH, conditions such as the condition of water washing complex and sulfuric acid solution adjust pH are investigated, determine that best purification condition is that extracting solution is through ethanol precipitation to 80%, centrifugal liquid adds 10% calcium oxide breast and transfers pH to 10, centrifugal, after the precipitation water washing once, be transferred in 90% ethanol, add 50% sulphuric acid and transfer pH to be 5, centrifugal, it is 6.5 that centrifugal liquid adds 40% sodium hydroxide accent pH, is the optimum condition of purification.
In the development, the preparation technology of the existing medicine DIEMAILING ZHUSHEYE in market carried out investigating and perfect, the innovative point of new technology is 1. to have increased the ethanol precipitation removal of impurity, has avoided adopting in the former technology unfavorable factor of remove impurity with active carbon matter.2. having increased water washing calcium oxide complex is to guarantee that effective site content reaches one step of key of injection requirement.Refining Herba Ixeritis Sonchifoliae total flavones content through trial-production can reach more than 80%, every inspection index meets the requirement of injection intravenously administrable, and carried out the pharmacodynamics contrast experiment with DIEMAILING ZHUSHEYE isodose medicinal substances extract, the result shows that the activity of refining Herba Ixeritis Sonchifoliae total flavones is apparently higher than DIEMAILING ZHUSHEYE.This technological design is reasonable, has novelty and production practicality.
(4) research of moulding process
The Chinese medicine ingredients complexity, easily degraded in aqueous solution, and microorganism is grown in aqueous solution easily, so, the stability of Chinese medicine is more unmanageable problem in the injection, it can directly influence the quality and the curative effect of product, so select for use suitable dosage form can avoid the appearance of these problems, guarantees the safe and reliable and stable of product.
(1) selection of freeze-dried powder dosage form
Because this product loading amount when the 20mg/ bottle, just reaches effective dose, so, on the loading amount of peace bottle, certain difficulty is arranged, be not easy to directly make injectable powder, so selected freeze-dried powder dosage form if select the injectable powder type.
(2) prescription is selected test
The clinical consumption of the plan of injection Herba Ixeritis Sonchifoliae is each 40mg, for ease of clinical practice, determine every powder charge 20mg, the injection Herba Ixeritis Sonchifoliae is made into 0.5,1.0,2.0% concentration respectively, selects the proppant of variety classes and different amounts for use, divide respectively to install in the 10ml cillin bottle, use the freezer dryer lyophilization, process conditions are at-40 ℃ of pre-freeze 2-3 hours, make fully and freeze, under about 11Pa vacuum by per hour 2 ℃ be warming up to-20 ℃; After distilling about 12 hours, in 8 hours, be warming up to 30 ℃, the results are shown in Table 1.
Table 1 prescription screening result
The result shows, 0.5% and 2% liquor strength is all undesirable to its shape of the prepared finished product of different amounts of two kinds of proppant selecting for use.0.5% concentration is easy-formation or easily fragmentation of evacuation not, and the finished product that 2% concentration makes is too thin, an easily sticking bottle end.And when selecting 1% liquor strength for use, add 3% or 4% mannitol to make proppant, no matter aspects such as color and luster, shape, post thickness, the close property of matter are the best, zero difference, and the finished product of 1% or 2% mannitol shows slightly evacuation.The every index of the finished product of gained did not have the mannitol ideal when Dextran-20 was made proppant by contrast, was prone to crackle after the molding.The above results determines that through analysis-by-synthesis proppant is selected 3% mannitol for use, and liquor strength is 1% o'clock, and the outward appearance of finished product is ideal.So preparation prescription is for being defined as: refining Herba Ixeritis Sonchifoliae total flavones 20g, mannitol 60g add the injection water to 2000ml, and every contains refining Herba Ixeritis Sonchifoliae total flavones 20mg.Prove that by lab scale prescription of this technology and lyophilization condition are feasible, formed product well meets the pertinent regulations of injection, for pilot scale provides certain technical basis.
Stability test research
Behind the process stabilizing, preparation has been carried out the preliminarily stabilised test, test is with intending the sample that listing is packed.Room temperature was placed after 3 months, and character, the solubility property of preparation all do not change; Three identifications all are positive; Check and all meet the requirements; Assay general flavone content and luteolin-7-O-beta d glucopyranosiduronic acid glycosides all meets the requirements; Room temperature is placed and was not found that a certain index has obvious change, showed that this product is more stable in three months in a word.
The formulation of injection Herba Ixeritis Sonchifoliae discrimination method of the present invention.
[discriminating] gets this product 20mg, is dissolved in the 5ml water, adds ethyl acetate 10ml and extracts, and ethyl acetate liquid, evaporate to dryness, residue add 60% ethanol 1ml makes dissolving, as need testing solution; Other gets luteolin 7-O-β-D-pyranglucoside reference substance, adds 60% ethanol and makes the solution that every 1ml contains 2mg, in contrast product solution.Test according to thin layer chromatography (appendix VIB of Chinese Pharmacopoeia version in 2005), drawing each 10 μ l of above-mentioned two kinds of solution, put respectively on the efficient lamellae of same silica gel G, is developing solvent with chloroform-methanol-glacial acetic acid (70: 20: 10), launch, take out, dry, spray is with 10% aluminum chloride alcoholic solution, put under the uviol 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.，
The formulation of finger printing
The method that the Chinese medicine fingerprint institute adopts roughly is divided into chromatography, spectrographic method.Chromatography comprises traditional thin layer chromatography, liquid chromatography.In order better to control the inherent quality of this product, adopt two kinds of chromatographic processes that the finger printing of raw medicinal material is investigated.
Although thin layer chromatography is traditional qualitative, quantitative analysis method, because of the sample composition complexity, all can't reach under four kinds of development system conditions fully and separate, some composition stack is serious, so this method of proof is inadvisable.Easy and simple to handle, characteristics such as separation efficiency is high, selectivity is high, detection sensitivity is high, analysis speed is fast, applied range that high performance liquid chromatography has.And the application experience of existing decades in this field, the finger printing research method should be selected according to the physicochemical properties of object of study, with the Herba Ixeritis Sonchifoliae is that raw material extracts purified refining Herba Ixeritis Sonchifoliae total flavones, general flavone content reaches more than 90%, flavone compound has very strong absworption peak under ultraviolet wavelength, can reach good separating degree by high performance liquid chromatography.Detect wavelength and can pass through the DAD detector, the relatively more different chromatographic peaks that detect under the wavelength are selected baseline to detect wavelength stably, and are selected the moderate detection wavelength of fingerprint peaks in conjunction with contained active ingredient.So the finger printing of this product is selected high performance liquid chromatography for use, and its condition is investigated, and aspects such as its mobile phase ratio, column type, column temperature, flow velocity, sample size are investigated, and has finally determined high-efficient liquid phase chromatogram condition.By methodological study, this method is feasible, has set up the standard finger-print of said preparation, has reached effective control product quality, guarantees the purpose of product stability.
Finger printing is measured in conjunction with State Food and Drug Administration's " specification requirement of Chinese medicine finger printing research " according to high performance liquid chromatography (an appendix VI of Pharmacopoeia of the People's Republic of China version in 2005 D).
Chromatographic condition and system suitability test
With octadecylsilane chemically bonded silica is filler; Mobile phase A is an acetonitrile, and B is 0.4% phosphoric acid solution (ml/ml), and the according to the form below program is carried out gradient elution; Flow velocity is 0.9ml/min; Column temperature is 35 ℃; The detection wavelength is 348nm.Number of theoretical plate calculates by luteolin-7-O-β-D-glucuronide peak should be not less than 5000.
The preparation of object of reference solution
It is an amount of that precision takes by weighing luteolin-7-O-beta d glucopyranosiduronic acid glycosides reference substance, adds 60% ethanol and make the solution that every 1ml contains 0.25mg, promptly.(ultrasonic in case of necessity 5 minutes)
The preparation of need testing solution
Precision takes by weighing refining Herba Ixeritis Sonchifoliae total flavones 60mg, is dissolved in water and is diluted in the 10ml measuring bottle, as need testing solution.
Algoscopy
Accurate respectively object of reference solution and each 5 μ l of need testing solution of drawing inject hplc determination respectively, write down the retention time and the integral area at each peak in 40 minutes, and detection threshold (smallest peaks area) is set at 1.0% of total peak area.8 total peaks should be arranged, and are with reference to peak S with No. 9 peaks, calculate each fingerprint peaks relative area and relative retention time, and the non-total peak gross area accounts for the percentage ratio of total peak area should be less than 5%, and its testing result is answered conformance with standard fingerprint pattern technology parameter.
Refining Herba Ixeritis Sonchifoliae total flavones standard finger-print and technical parameter
Description of drawings:
The standard finger-print of Fig. 1-----luteolin-7-O-beta d glucopyranosiduronic acid glycosides reference substance.
The standard finger-print technical parameter
Attached instrument: LC-2010 high performance liquid chromatograph, CLASS-VP work station. Chromatographic column: Diamonsil C18(the chromatography post of 4.6mm * 250mm).
The pH value is got this product 20mg, injects water 50ml and makes dissolving, measures (middle traditional Chinese medicines in accordance with the law An appendix VII of allusion quotation version in 2005 G), should be 4.0～5.5.
Sulfate is got this product 20mg, injects water 10ml and makes dissolving, gets 2ml, checks in accordance with the law (two appendix VIII of Chinese Pharmacopoeia version in 2005 B), the contrast liquor ratio made from standard potassium sulfate solution 1ml , must not denseer (0.005%).
Calcium salt is got this product 20mg, injects water 10ml and makes dissolving, gets 2ml, adds the ammonium oxalate examination Liquid 1ml shakes up, and places 10 minutes, must not show muddy.
Moisture is got this product, according to an aquametry (appendix IX of Chinese Pharmacopoeia version in 2005 H first Method) measures, must not cross 5.0%.
Visible foreign matters is got 5 parts of this product, and every part of 20mg injects respectively water 10ml and makes dissolving, Check according to State Food and Drug Administration's " visible foreign matters inspection technique supplementary provisions ", should meet rule Fixed.
Protein is got this product 20mg, injects water 10ml and makes dissolving, gets 1ml, adds the tannic acid test solution 1-3 drips, and muddiness must not appear in mixing.
Tannin is got this product 20mg, injects water 10ml and makes dissolving, get 1ml, adds new preparation The physiological saline 5mL that contains 1% egg behind the mixing, placed 10 minutes. and muddiness or precipitation must not appear.
Resin is got this product 15mg, injects water 5ml and makes dissolving, adds chloroform 10ml, and jolting is extracted, Divide and get chloroform solution, evaporate to dryness, residue add glacial acetic acid 2ml makes dissolving, puts in the tool plug test tube, adds water 3ml, mixing, Placed 30 minutes, and should not have precipitation and occur.
Residue on ignition is got this product 1.0g, checks (an appendix IX of Chinese Pharmacopoeia version in 2005 J) in accordance with the law, Must not cross 15%.
Oxalates is got this product 20mg, injects water 10ml and makes dissolving, gets 2ml, check (in accordance with the law An appendix IX of state's pharmacopeia version in 2005 S), muddiness or precipitation must not appear.
Potassium ion is got this product 20mg, injects water 10ml and makes dissolving, checks (Chinese Pharmacopoeia in accordance with the law An appendix IX of version in 2005 S), the contrast liquor ratio of in accordance with the law making with standard potassium ion solution 0.8ml, Must not be denseer.
Bacterial endotoxin is got this product, checks (two appendix XI of Chinese Pharmacopoeia version in 2005 E) in accordance with the law, Every 1mg contains endotoxic amount should be less than 1.25EU.
The foundation of the content assaying method of general flavone in the sowthistle-leaf ixeris seedling powder ampoule agent for injection of the present invention. Adopt warp Allusion quotation method alkali-natrium nitrosum-aluminum nitrate colorimetric method is measured method to refining general ixeris flavone Easy, reliable results have adopted the method in the standard. Select simultaneously rutin, cyanidenon-7-O-β-D-Phranoglucuronide, cyanidenon-7-O-β-three reference substances of D-glucopyranoside carry out respectively The investigation of determination of total flavonoids, three reference substance measurement results are more approaching, because of in this product with reseda Element-7-O-beta d glucopyranosiduronic acid glycosides content is higher, so select cyanidenon-7-O-β-D-pyrans Portugal The grape glycuronide is reference substance.
The content assaying method of general flavone is in the sowthistle-leaf ixeris seedling powder ampoule agent for injection of the present invention:
The preparation precision of reference substance solution takes by weighing at 105 ℃ of drying under reduced pressure to the constant weight cyanidenon The reference substance of 7-O-beta d glucopyranosiduronic acid glycosides is an amount of, adds 60% ethanol and makes every 1ml and contain 0.1mg The solution of cyanidenon 7-O-beta d glucopyranosiduronic acid glycosides (in case of necessity, can be ultrasonic 5 minutes, make Dissolve) and get final product.
Accurate reference substance solution 1.0,2.0,3.0,4.0, the 5.0ml of drawing of the preparation of calibration curve, Put respectively in the 10ml measuring bottle, each adds 5% sodium nitrite solution 0.3ml, shakes up, and places 6 minutes. Add respectively 10% aluminum nitrate solution 0.3ml, shake up, placed 6 minutes, add again the 1mol/L hydrogen-oxygen Change sodium solution 4ml, be diluted to scale with 60% ethanol, shake up, placed 10 minutes. According to spectrophotometric Method (appendix VA of Chinese Pharmacopoeia version in 2005) is measured trap at 505nm wavelength place, with Trap is ordinate, take concentration as abscissa, and the drawing standard curve.
The determination method precision takes by weighing solids 5mg, puts in the 50ml measuring bottle, adds 60% ethanol and is diluted to Scale shakes up, as need testing solution. The accurate need testing solution 5ml that draws puts in the 10ml measuring bottle, Method under the sighting target directrix curve preparation from " adding 5% sodium nitrite solution 0.3ml ", is surveyed in accordance with the law Decide trap, get simultaneously need testing solution 5ml, add 60% ethanol and be diluted to 10ml, as blank correction, Calculate, and get final product.
This product contains general flavone with cyanidenon-7-O-beta d glucopyranosiduronic acid glycosides (C21H
18O
12) meter, Must not be less than 80.00%.
In order further to control the quality of product, the cyanidenon higher to content in the sample-7-O-β-D-The phranoglucuronide content measuring standard is formulated, and method adopts finger-print wash-out bar Part. Cyanidenon in the sowthistle-leaf ixeris seedling powder ampoule agent for injection of the present invention-7-O-beta d glucopyranosiduronic acid glycosides Content assaying method is:
Measure according to high performance liquid chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 D).
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler; Flow Phase A is acetonitrile, and B is 0.4% phosphoric acid solution (ml/ml), and the according to the form below program is carried out gradient elution; Column temperature is 35 ℃; The detection wavelength is 348nm. Number of theoretical plate is pressed cyanidenon-7-O-β-D-pyrans grape The glycuronide peak calculates should be not less than 5000.
The preparation precision of reference substance solution takes by weighing at the 105 ℃ of cyanidenon that is dried to constant weight-7-O-β-D-pyrroles Glucopyranoside aldehydic acid glycosides reference substance is an amount of, and add 60% ethanol and make the solution that every 1ml contains 0.25mg, And get final product. (in case of necessity, can make dissolving in ultrasonic 5 minutes)
The preparation precision of need testing solution takes by weighing solids 60mg, puts in the 10ml measuring bottle, and water dissolves also Be diluted to scale, shake up, and get final product.
Determination method is accurate reference substance solution and each 5 μ l of need testing solution of drawing respectively, and the injection liquid chromatography is measured, and this product contains cyanidenon-7-O-beta d glucopyranosiduronic acid glycosides (C21H
18O
12) must not be less than 8.00%.
The preparation of embodiment 1 Herba Ixeritis Sonchifoliae total flavones
Get the Herba Ixeritis Sonchifoliae medical material, decoct with water secondary, add 12 times of water gagings for the first time, decocted 1 hour, add 10 times of water gagings for the second time, decocted 1 hour; Collecting decoction is concentrated into every 1ml and is equivalent to crude drug in whole 1g, puts cold, stir down and add ethanol, make to contain alcohol and measure and reach 80%, left standstill 12 hours, decompression filters, and filtrate adds 10% calcium oxide breast under stirring, and regulates pH value to 11.0, leave standstill 0.5 hour, centrifugal, precipitate adds 10 times of water gagings, stirred 10 minutes, centrifugal, precipitate adds ethanol to be made and contains alcohol amount and reach 90%, adds 50% sulfuric acid solution under stirring, regulate pH value to 5.0, centrifugal, centrifugal liquid adds 40% sodium hydroxide solution under stirring, and regulates pH value to 6.5, filter, decompression filtrate recycling ethanol adds the injection water and is dissolved to 100ml, freezing 24 hours to there not being the alcohol flavor, room temperature is melted, decompression filters, and the filtrate lyophilization gets Herba Ixeritis Sonchifoliae total flavones.
The preparation of embodiment 2 Ixers sonchifolia freeze-dried powder for injection
Get Herba Ixeritis Sonchifoliae total flavones 20g; mannitol 60g adds the injection water and makes dissolving in right amount, 100 ℃ of steam sterilizations 30 minutes; add the injection water and be settled to 2000ml, decompression filters, and filtrate is aseptic subpackaged in cillin bottle; put in the drying baker; freezed 2-3 hour in-40 ℃, reach at condenser below-45 ℃, open vacuum pump; open drying baker and condenser butterfly valve; reach 11Pa approximately in about 30 minutes inner drying case vacuum, heated up 2 ℃, be warming up to-20 ℃ by average about 1 hour; distilled altogether 12 hours; by per hour heating up 8 ℃, closely consistent with baffle temperature again to goods, shut down; lyophilization; close plug, gland is made 1000.
The Ixers sonchifolia freeze-dried powder for injection that makes is hereinafter to be referred as the injection Herba Ixeritis Sonchifoliae, and content is light brown yellow stopper column, the about 16mm of diameter, and the about 7mm of thickness, quality is even, and molding is good.
In the injection Herba Ixeritis Sonchifoliae injectable powder of the present invention, heavy dose of group all can obviously dwindle rat coronary ligation MIS after 24 hours, reduce serum CK, AST and LDH activity, reduce platelet adhesion and aggregation, also can obviously reduce Serum LPO content, improve SOD and GSH-Px activity; In the injection Herba Ixeritis Sonchifoliae, heavy dose of group all can obviously alleviate the rat ischemia ECG change that pituitrin brings out; In the injection Herba Ixeritis Sonchifoliae, heavy dose of group all can obviously dwindle myocardial ischemia reperfusion injury rat MIS, reduce serum AST, CK and LDH activity, reduce Serum LPO content, improve SOD in serum and GSH-Px activity, and can obviously reduce myocardial infarction district and non-infarcted region FFA level, reduce rat plasma ET, AngII and TXA2 level, increase PGI2 level and PGI2/TXA2 ratio; In the injection Herba Ixeritis Sonchifoliae, heavy dose of group all can obviously alleviate AMI dog degree of myocardial ischemia, reduces the myocardial ischemia scope, dwindles MIS, obviously reduces serum AST, CK, LDH activity; In the injection Herba Ixeritis Sonchifoliae, heavy dose of group all can obviously resist the myocardial oxygen consumption that the subcutaneous injection isoproterenol causes, prolongs the time-to-live of mice under the normobaric hypoxia state; Blood pressure (SBP, DBP and MAP) in the injection Herba Ixeritis Sonchifoliae, when heavy dose of group all can obviously reduce acute myocardial infarction reduces, and obviously increase LVP, ± dp/dtmax, CO and CI, explanation can improve myocardium systolic and diastolic function, help to resist pump failure after the infarction, simultaneously can obviously reduce AMI dog LVEDP, can alleviate cardiac preload, reduce cardiac work, show that the injection Herba Ixeritis Sonchifoliae can obviously improve pumping function behind the myocardial infarction; The injection Herba Ixeritis Sonchifoliae can obviously increase myocardial flow after the infarction, and showing to increase blood supply of cardiac muscle; In the injection Herba Ixeritis Sonchifoliae, heavy dose of group can suppress that the rat thrombus in vivo forms and external thrombus forms; In the injection Herba Ixeritis Sonchifoliae, heavy dose of group all can obviously reduce the whole blood of stasis syndrome rat model lowly cut, in cut, height is cut viscosity and plasma viscosity, and obviously reduces plasma fibrinogen concentration, erythrocyte electrophoretic time, erythrocyte sedimentation rate and packed cell volume.
The above results shows that injection Herba Ixeritis Sonchifoliae treatment acute myocardial ischemia has good curative effect, and its potency is apparently higher than KUDIEZI ZHUSHEYE.
The influence of 1 pair of rat coronary ligation of experimental example acute myocardial infarction (AMI) model
Test drug: injection Herba Ixeritis Sonchifoliae, specification: contain refining Herba Ixeritis Sonchifoliae total flavones 20mg/ and prop up (be equivalent to 10g crude drug /), provide by the preparation of embodiment 2 methods by Tonghua Huaxia Pharmaceutical Co., Ltd..Be formulated as desired concn with sodium chloride injection during test, dosage calculates to contain the crude drug amount.
The negative control medicine: sodium chloride injection, the sincere pharmaceutcal corporation, Ltd in sky, Changchun produces lot number: 20020810.
Positive control drug: Herba Ixeritis Sonchifoliae (spirit of dish arteries and veins) injection, specification: contain total flavones 6mg/10ml and (be equivalent to 10g crude drug/lot number 10ml), is provided by Tonghua Huaxia Pharmaceutical Co., Ltd.: 20021205.Be formulated as desired concn with sodium chloride injection during test, dosage calculates to contain the crude drug amount.
Animal
The Wistar rat, body weight 260～300g, the quality certification number: the moving word 10-5110 of doctor, male and female half and half are provided by Changchun High-technology Medical Animal Experiment Research Center.
Test method
90 rats are divided into 6 groups at random by body weight, that is: sham-operation (sodium chloride injection) group, infarction model (sodium chloride injection) group, positive control drug KUDIEZI ZHUSHEYE 10g crude drug/kg (be equivalent to clinical day medication 40g crude drug/60kg people/sky 2.8 times) group and injection Herba Ixeritis Sonchifoliae 1.25,2.5,5g crude drug/kg (be equivalent to clinical day medication 20g crude drug/60kg people/sky 0.7,1.4,2.8 times) organize 15 every group.Press literature method and make myocardial infarction model
[1]: under etherization, rat is faced upward the position be fixed on the operating-table, measure rat II and lead behind the normal ECG, 3～4 intercostals are opened breast from the left side, expose heart, in between pulmonary conus and left atrium, find out the arteria coronaria left anterior descending branch, with No. 0 line ligation immediately it, send heart back to thoracic cavity, and extrude thoracic cavity inner blood and gas, close the thoracic cavity rapidly, wholely open the breast time and be no more than 30 seconds, not ligation of sham operated rats and only put surgical thread.Each treated animal is distinguished sublingual vein drug administration by injection 1 time with 6h after surgery immediately.After treating ligation arteria coronaria 24h,, measure rat II lead electrocardiogram with pentobarbital sodium 30mg/kg intraperitoneal injection of anesthesia.Then, abdominal aortic cannulation is got blood, survey serum aspartic acid aminotransferase (AST), creatine phosphokinase (CPK) and lactic acid dehydrogenase (LDH) activity, and by test kit (bio-engineering research institute is built up in Nanjing) survey serum lipid peroxide (LPO) content, erythrocuprein (SOD) and glutathion peroxidase (GSH-Px) activity.From abdominal aortic blood 1ml,, to get 10 μ l and add in the 1ml platelet diluent in ratio anticoagulant in 1: 9 with 3.8% sodium citrate, the shake mixing splashes into cell counting count board, leaves standstill 10min, platelet Counting number under optical microscope.Residue blood adds in the LBY-F5 platelet adhesion instrument ball, behind the rotation 15min, and the platelet count after counting adheres under light microscopic, { (adhering to thromboblast number-adhesion back platelet count)/adhesion thromboblast number * 100%} calculates platelet adhesion rate according to formula
[2]From abdominal aortic blood 3ml, with 3.8% sodium citrate anticoagulant mixing, with the centrifugal 5min of 180 commentaries on classics/min, getting supernatant was platelet rich plasma (PRP), puts into 0.5ml doffer pipe, puts in the constant temperature hole standby in 1: 9 ratio; Remaining anticoagulation is with the centrifugal 10min of 300 commentaries on classics/min, the gained supernatant is platelet poor plasma (PPP), respectively get PPP, PRP 200 μ l, adopt LBY-NJ2 platelet aggregation instrument (Beijing Puli gives birth to group and produces) to measure platelet aggregation, obtain 1,3 and platelet aggregation rate (PAG) and the maximum platelet aggregation rate (MPAG) of 5min.Take out rat heart rapidly,, remove atrium and fatty tissue and weigh with normal saline flush away chambers of the heart inner blood.With 4～5 of ventricle crosscuts, put in chlorination nitro blue tetrazolium (NB-T) phosphate buffer, take out the back fully in 37 ℃ of water bath with thermostatic control dyeing.Normal structure dyeing, ischemic tissue does not dye.Downcut ischemic tissue and weigh, with the percentage calculation myocardial infarct size (MIS) of ischemic myocardium and ventricle weight in wet base.Test data is represented with means standard deviation, by t check carrying out statistical analysis between group.
Result of the test
The influence of the 1 couple of AMI rat MIS and serum AST, CK and LDH
Compare with sham operated rats, the MIS of infarction model group rat and serum AST, CK, LDH activity all obviously increase (p＜0.01 or p＜0.001).With the infarction model group relatively, in the injection Herba Ixeritis Sonchifoliae, heavy dose of group all can obviously dwindle rat coronary ligation MIS (p＜0.05 or p＜0.01) after 24 hours, and can obviously reduce serum AST, CK and LDH activity (p＜0.05 or p＜0.01), see Table 1.
Table 1. injection Herba Ixeritis Sonchifoliae to the protective effect of rat acute myocardial infarction (x ± s, n=10)
Compare ##p＜0.01, ###p＜0.001 with sham operated rats; Compare * p＜0.05, * * p＜0.01 with the infarction model group
The influence of 2 couples of AMI rat blood serum LPO, SOD and GSH-Px
Compare with sham operated rats, infarction model group rat blood serum LPO content obviously increases, active obviously reduce (p＜0.05 or p＜0.01) of SOD and GSH-Px.With the infarction model group relatively, in the injection Herba Ixeritis Sonchifoliae, heavy dose of group all can obviously reduce rat coronary ligation Serum LPO content (p＜0.05 or p＜0.01) after 24 hours, also can obviously improve SOD in serum and GSH-Px activity (p＜0.05), sees Table 2.
Table 2. injection Herba Ixeritis Sonchifoliae to the influence of AMI rat blood serum LPO, SOD and GSH-Px (x ± s, n=10)
Compare #p＜0.05, ##p＜0.01 with sham operated rats; Compare * p＜0.05 with the infarction model group, material p＜0.01
3 pairs of AMI rat platelets stick the influence of function
(%, x ± s be 44.51 ± 9.52 n=10), and the platelet adhesion reaction rate of infarction model group are 63.84 ± 11.84 to the platelet adhesion reaction rate of sham operated rats, apparently higher than sham operated rats (p＜0.01).The injection Herba Ixeritis Sonchifoliae is little, in, the platelet adhesion reaction rate of heavy dose of group is respectively 57.30 ± 14.02,50.15 ± 9.30 and 46.71 ± 7.86, compare with the infarction model group, in the injection Herba Ixeritis Sonchifoliae, heavy dose of group all can obviously reduce platelet adhesion reaction rate (p＜0.05 or p＜0.01), small dose group does not have obvious influence (p＞0.05) to the platelet adhesion reaction rate.The platelet adhesion reaction rate of KUDIEZI ZHUSHEYE is 51.83 ± 9.44, with infarction model group comparing difference remarkable (p＜0.05).
The influence of 4 pairs of AMI rat platelet aggregation functions
With sham operated rats relatively, infarction model group rat 1,3,5min and maximum platelet aggregation rate all obviously increase (p＜0.05～p＜0.001).In the injection Herba Ixeritis Sonchifoliae, heavy dose of group can reduce obviously all that ADP is inductive 1,3,5min and maximum platelet aggregation rate, has significance (p＜0.05～p＜0.001) with infarction model group comparing difference, sees Table 3.
Table 3. injection Herba Ixeritis Sonchifoliae to the influence of AMI rat platelet aggregation function (x ± s, n=10)
Compare #p＜0.05, ##p＜0.01, ###p＜0.001 with sham operated rats;
Compare * p＜0.05, * * p＜0.01, * * * p＜0.001 with the infarction model group
2 pairs of pituitrin of experimental example bring out the Electrocardiographic influence of Acute Myocardial Ischemia in Rats
Medicine
Test drug: injection Herba Ixeritis Sonchifoliae, specification: contain refining Herba Ixeritis Sonchifoliae total flavones 20mg/ and prop up (be equivalent to 10g crude drug /), provide by the preparation of embodiment 2 methods by Tonghua Huaxia Pharmaceutical Co., Ltd..Be formulated as desired concn with sodium chloride injection during test, dosage calculates to contain the crude drug amount.
The negative control medicine: sodium chloride injection, the sincere pharmaceutcal corporation, Ltd in sky, Changchun produces lot number: 20020810.
Positive control drug: Herba Ixeritis Sonchifoliae (spirit of dish arteries and veins) injection, specification: contain total flavones 6mg/10ml and (be equivalent to 10g crude drug/lot number 10ml), is provided by Tonghua Huaxia Pharmaceutical Co., Ltd.: 20021012.Be formulated as desired concn with sodium chloride injection during test, dosage calculates to contain the crude drug amount.
Animal
The Wistar rat, body weight 180～220g, the quality certification number: the moving word 10-5110 of doctor, male and female half and half are provided by Changchun High-technology Medical Animal Experiment Research Center.
Test method
Careful electric sieve selects 50 of qualified rats to be divided into 5 groups at random, that is: contrast (sodium chloride injection) group, positive control drug KUDIEZI ZHUSHEYE 10g crude drug/kg (be equivalent to clinical day medication 40g crude drug/60kg people/sky 2.8 times) group and injection Herba Ixeritis Sonchifoliae 1.25,2.5,5g crude drug/kg (be equivalent to clinical day medication 20g crude drug/60kg people/sky 0.7,1.4,2.8 times) organize 10 every group.Each organizes equal sublingual vein drug administration by injection, every day 1 time, continuous 3 days.30min behind the last medicine from sublingual vein injection of pituitrin (Shanghai Hefeng Pharmaceutical Co., Ltd.'s product, lot number: 020801, dilute with normal saline during test) 1u/kg, injected in 10 seconds and finishes, and traced II lead electrocardiogram in the 10min.Measure to give that ST section and T ripple change in the pituitrin 30 seconds.Test data is by t check carrying out statistical analysis between group.
Result of the test
Visible II lead electrocardiogram ST section and T ripple are all obviously raised behind the matched group sublingual vein injection of pituitrin.In the injection Herba Ixeritis Sonchifoliae, heavy dose of group all can obviously alleviate Acute Myocardial Ischemia in Rats electrocardiogram (the ST section is raised and the T wave height is alarmmed) intensity of variation that the sublingual vein injection of pituitrin brings out, with matched group comparing difference remarkable (p＜0.01 or p＜0.001), point out it can alleviate acute myocardial ischemia due to the coronary spasm, see Table 4.
Table 4. injection Herba Ixeritis Sonchifoliae to pituitrin bring out the Electrocardiographic influence of rat ischemia (x ± s, n=10)
Compare * * p＜0.01, * * * p＜0.001 with matched group
Experimental example 3, to the influence of dog coronary ligation acute myocardial infarction (AMI) model
Medicine
Test drug: injection Herba Ixeritis Sonchifoliae, specification: contain refining Herba Ixeritis Sonchifoliae total flavones 20mg/ and prop up (be equivalent to 10g crude drug /), provide lot number by Tonghua Huaxia Pharmaceutical Co., Ltd. by the preparation of embodiment 2 methods: 20030120.Be formulated as desired concn with sodium chloride injection during test, dosage calculates to contain the crude drug amount.
The negative control medicine: sodium chloride injection, the sincere pharmaceutcal corporation, Ltd in sky, Changchun produces lot number: 20020810.
Positive control drug: Herba Ixeritis Sonchifoliae (spirit of dish arteries and veins) injection, specification: contain total flavones 6mg/10ml and (be equivalent to 10g crude drug/lot number 10ml), is provided by Tonghua Huaxia Pharmaceutical Co., Ltd.: 20021012.Be formulated as desired concn with sodium chloride injection during test, dosage calculates to contain the crude drug amount.
Animal
Healthy hybrid dog, the male and female dual-purpose, body weight 12.5～14.5kg, Jilin University's Experimental Animal Center (former Norman Bethune Medical University laboratory animal portion) provides.
Test method
30 of healthy adult hybrid dogs are divided into 5 groups at random, that is: model contrast (sodium chloride injection) group, positive control drug KUDIEZI ZHUSHEYE 4g crude drug/kg (be equivalent to clinical day medication 40g crude drug/60kg people/sky 3.8 times) group and injection Herba Ixeritis Sonchifoliae 0.5,1,2g crude drug/kg (be equivalent to clinical day medication 20g crude drug/60kg people/sky 0.9,1.9,3.8 times) organize 6 every group.Anaesthetize with pentobarbital sodium 30mg/kg intravenous injection during test.Cervical region tracheostomize intubate connects artificial respirator and keeps ventilation.The 4th intercostal is opened breast in a left side, separate a blunt edge that circles round on an arteria coronaria left side and prop up, LAD the 1st, 2 branches and prop up with each side of blunt edge Zhi Xianglian and ramus anastomoticus in order to ligation, make the predetermined approximately quite left chamber of ischemic region 1/2 of the wall front surface that dissociates.With the wet cloth formula absorption method mapping epicardial electrogram (EECG) of multiple spot, 24 of mapping points divide normally district, infarction marginal zone and infarction center.Art finishes, and stablize 15min, writes down each point EECG and is worth before as administration.Each group is all executed coronary ligation.5min after ligation is dissolved among the sodium chloride injection 200ml medicinal liquid in right lateral thigh vein constant speed venoclysis 90min.Before ligation and administration after 5,10,15,30,60,90,120,180,240,300,360min mapping epicardial electrogram.Use the moistening heart surface of normal saline before each mapping.Demarcate normal voltage, make the 10mm of recorder chart be equivalent to 5mv.Each mapping is put the summation (∑ ST) that mv value that average ST section raises and ST section raise in the experiment with computing process, and the ∑ ST difference DELTA ∑ ST before and after the ligation, the degree of expression myocardial ischemia.The electrodeplate that the rising of ST section is equal to or greater than 2mv is NST, and the NST difference before and after the ligation is Δ NST, expression myocardial ischemia scope
[6]360min gets blood from femoral artery after the ligation, surveys serum aspartic acid aminotransferase (AST), creatine phosphokinase (CK) and lactic acid dehydrogenase (LDH) activity with the COBAS-FAARA automatic biochemistry analyzer; It is dirty to core simultaneously, removes the atrium, takes by weighing the ventricle weight in wet base.Be parallel to coronary sulcus with 4～5 of ventricle crosscuts, carry out N-BT dyeing.Treat to take out immediately when the infarcted myocardium boundary line is known, cut infarcted myocardium and weigh.With the percentage ratio of infarcted myocardium and ventricle weight in wet base as myocardial infarction area (MIS).Test data is by t check carrying out statistical analysis between group.
Result of the test
The influence of 1 pair of AMI dog degree of myocardial ischemia (Δ ∑ ST) and ischemia scope (Δ NST)
The all visible Δ ∑ ST of infarction matched group 30min～360min behind medicine obviously increases, with comparing difference before the medicine remarkable (p＜0.001).Compare with the infarction matched group, injection Herba Ixeritis Sonchifoliae small dose group behind medicine 30,60,90min can obviously reduce Δ ∑ ST (p＜0.05), in the dosage group behind medicine 30,60,90,120,180,240min can obviously reduce Δ ∑ ST (p＜0.05 or 0.01), heavy dose organizes behind medicine 30,60,90,120,180,240,300,360min all can obviously reduce Δ ∑ ST (p＜0.05 or 0.01), sees Table 10; In the injection Herba Ixeritis Sonchifoliae dosage group behind medicine 60,90,120,180,240min can obviously reduce Δ NST, with infarction matched group comparing difference remarkable (p＜0.05 or 0.01), injection Herba Ixeritis Sonchifoliae heavy dose organizes behind medicine 30,60,90,120,180,240,300min can obviously reduce Δ NST, with infarction matched group comparing difference significantly (p＜0.05 or 0.01).
The influence of the 2 couples of AMI dog MIS and serum cardiac muscle three enzymatic activitys
Compare with the infarction matched group, three dosage groups of injection Herba Ixeritis Sonchifoliae all can obviously be dwindled AMI dog MIS (P＜0.05～P＜0.001); In the injection Herba Ixeritis Sonchifoliae, heavy dose of group also can obviously reduce acute myocardial infarction dog serum LDH, AST and CK activity (P＜0.05 or 0.01).
Claims (9)
1, Herba Ixeritis Sonchifoliae total flavones, it is prepared by following method: get the Herba Ixeritis Sonchifoliae medical material and decoct with water, with decocting liquid, be concentrated into every 1ml and be equivalent to crude drug in whole 1g, put coldly, add ethanol under stirring, make to contain alcohol amount and reach 80%, leave standstill, filter, add alkali under filtrate is stirred and regulate pH value to 11, leave standstill, centrifugal, precipitate adds 10 times of water gagings, stirs, and is centrifugal, precipitate adds ethanol to be made and contains alcohol amount and reach 90%, stirs down to add acid, adjusting pH value to 5, centrifugal, centrifugal liquid adds aqueous slkali under stirring, and regulates pH value to 6.5, filters, decompression filtrate recycling ethanol is to there not being the alcohol flavor, be dissolved in water, freezing, room temperature is melted, decompression filters, the filtrate lyophilization promptly gets Herba Ixeritis Sonchifoliae total flavones, wherein contains luteolin-7-O-beta d glucopyranosiduronic acid glycosides and is no less than 8.00%.
2, the described Herba Ixeritis Sonchifoliae total flavones of claim 1, it is prepared by following method: get the Herba Ixeritis Sonchifoliae medical material, decoct with water secondary, add 12 times of water gagings for the first time, decocted 1 hour, add 10 times of water gagings for the second time, decocted 1 hour; Collecting decoction is concentrated into every 1ml and is equivalent to crude drug in whole 1g, puts cold, stir down and add ethanol, make to contain alcohol and measure and reach 80%, left standstill 12 hours, decompression filters, and filtrate adds 10% calcium oxide breast under stirring, and regulates pH value to 11.0, leave standstill 0.5 hour, centrifugal, precipitate adds 10 times of water gagings, stirred 10 minutes, centrifugal, precipitate adds ethanol to be made and contains alcohol amount and reach 90%, adds 50% sulfuric acid solution under stirring, regulate pH value to 5.0, centrifugal, centrifugal liquid adds 40% sodium hydroxide solution under stirring, and regulates pH value to 6.5, filter, decompression filtrate recycling ethanol adds the injection water and is dissolved to 100ml, freezing 24 hours to there not being the alcohol flavor, room temperature is melted, decompression filters, and the filtrate lyophilization gets Herba Ixeritis Sonchifoliae total flavones.
3, the preparation method of the described Herba Ixeritis Sonchifoliae total flavones of claim 1, it comprises the following steps: to get the Herba Ixeritis Sonchifoliae medical material and decocts with water, with decocting liquid, be concentrated into every 1ml and be equivalent to crude drug in whole 1g, put coldly, add ethanol under stirring, make to contain alcohol amount and reach 80%, leave standstill, filter, add alkali under filtrate is stirred and regulate pH value to 11, leave standstill, centrifugal, precipitate adds 10 times of water gagings, stirs, and is centrifugal, precipitate adds ethanol to be made and contains alcohol amount and reach 90%, stirs down to add acid, adjusting pH value to 5, centrifugal, centrifugal liquid adds aqueous slkali under stirring, and regulates pH value to 6.5, filter, decompression filtrate recycling ethanol is dissolved in water to there not being the alcohol flavor, freezing, room temperature is melted, and decompression filters, the filtrate lyophilization promptly gets Herba Ixeritis Sonchifoliae total flavones.
4, the described Herba Ixeritis Sonchifoliae total flavones preparation method of claim 3, it comprises the following steps:
Get the Herba Ixeritis Sonchifoliae medical material, decoct with water secondary, add 12 times of water gagings for the first time, decocted 1 hour, add 10 times of water gagings for the second time, decocted 1 hour; Collecting decoction is concentrated into every 1ml and is equivalent to crude drug in whole 1g, puts cold, stir down and add ethanol, make to contain alcohol and measure and reach 80%, left standstill 12 hours, decompression filters, and filtrate adds 10% calcium oxide breast under stirring, and regulates pH value to 11.0, leave standstill 0.5 hour, centrifugal, precipitate adds 10 times of water gagings, stirred 10 minutes, centrifugal, precipitate adds ethanol to be made and contains alcohol amount and reach 90%, adds 50% sulfuric acid solution under stirring, regulate pH value to 5.0, centrifugal, centrifugal liquid adds 40% sodium hydroxide solution under stirring, and regulates pH value to 6.5, filter, decompression filtrate recycling ethanol adds the injection water and is dissolved to 100ml, freezing 24 hours to there not being the alcohol flavor, room temperature is melted, decompression filters, and the filtrate lyophilization gets Herba Ixeritis Sonchifoliae total flavones.
5. contain the injection freeze-dried powder that right requires 1 or 2 described Herba Ixeritis Sonchifoliae total flavoness, contain Herba Ixeritis Sonchifoliae total flavones and be no less than 80.0%.
6, the preparation method of the described Ixers sonchifolia freeze-dried powder for injection of claim 5, it comprises the following steps:
Get Herba Ixeritis Sonchifoliae total flavones 20g, mannitol 60g, add the injection water and make dissolving in right amount, 100 ℃ of steam sterilizations 30 minutes add the injection water and are settled to 2000ml, and decompression filters, and filtrate is aseptic subpackaged in cillin bottle, lyophilization, and close plug, gland is made 1000.
7, the detection method of the described Ixers sonchifolia freeze-dried powder for injection of claim 5, it comprises:
A. the preparation of object of reference solution
It is an amount of that precision takes by weighing luteolin-7-O-beta d glucopyranosiduronic acid glycosides reference substance, adds 60% ethanol and make the solution that every 1ml contains 0.25mg, promptly;
B. the preparation of need testing solution
Get 3 of injection Herba Ixeritis Sonchifoliaes, be dissolved in water and be diluted in the 10ml measuring bottle, as need testing solution;
C. algoscopy
With octadecylsilane chemically bonded silica is filler; Mobile phase A is an acetonitrile, and B is the 0.4%ml/ml phosphoric acid solution, and A by 31% and 69% B made the eluent eluting 40 minutes, makes the eluent eluting 40.01 minutes by 10% A and 90% B again; Flow velocity is 0.9ml/min; Column temperature is 35 ℃; Detecting wavelength is that the 348nm theoretical cam curve should be not less than 5000 by luteolin-7-O-β-D-glucuronide peak calculating; Accurate respectively object of reference solution and each 5 μ l of need testing solution of drawing inject hplc determination respectively, write down the retention time and the integral area at each peak in 40 minutes, and the threshold setting that detects the smallest peaks area is 1.0% of a total peak area; 8 total peaks should be arranged, and are with reference to peak S with No. 9 peaks, calculate each fingerprint peaks relative area and relative retention time, and the non-total peak gross area accounts for the percentage ratio of total peak area should be less than 5%, and its testing result is answered conformance with standard fingerprint pattern technology parameter, referring to Fig. 1:
8. the determination of total flavonoids method in claim 5 Ixers sonchifolia freeze-dried powder for injection, it comprises:
A) the preparation precision of reference substance solution takes by weighing at 105 ℃ of drying under reduced pressure in right amount to the reference substance of constant weight luteolin 7-O-beta d glucopyranosiduronic acid glycosides, adds 60% ethanol and makes the solution that every 1ml contains 0.1mg luteolin 7-O-beta d glucopyranosiduronic acid glycosides;
B) accurate reference substance solution 1.0,2.0,3.0,4.0, the 5.0ml of drawing of the preparation of standard curve puts respectively in the 10ml measuring bottle, and each adds 5% sodium nitrite solution 0.3ml, shakes up, and places 6 minutes.Add 10% aluminum nitrate solution 0.3ml respectively, shake up, placed 6 minutes, add 1mol/L sodium hydroxide solution 4ml again, to scale, shake up, placed 10 minutes with 60% ethanol dilution.Measuring trap at 505nm wavelength place according to spectrophotography, is vertical coordinate with the trap, is abscissa with concentration, the drawing standard curve;
C) the algoscopy precision takes by weighing solids 5mg, puts in the 50ml measuring bottle, adds 60% ethanol dilution to scale, shakes up, as need testing solution; The accurate need testing solution 5ml that draws, put in the 10ml measuring bottle, method under the sighting target directrix curve preparation, from " adding 5% sodium nitrite solution 0.3ml ", measure trap in accordance with the law, get need testing solution 5ml simultaneously, add 60% ethanol dilution to 10ml, as blank correction, calculate content of total flavone in this product, in luteolin-7-O-beta d glucopyranosiduronic acid glycosides.
9. the content assaying method of luteolin in claim 5 Ixers sonchifolia freeze-dried powder for injection-7-O-beta d glucopyranosiduronic acid glycosides, it comprises:
A) test of chromatographic condition and system suitability is a filler with octadecylsilane chemically bonded silica; Mobile phase A is an acetonitrile, and B is the 0.4%ml/ml phosphoric acid solution, and A by 31% and 69% B made the eluent eluting 40 minutes, makes the eluent eluting 40.01 minutes by 10% A and 90% B again; Flow velocity is 0.9ml/min; Column temperature is 35 ℃; The detection wavelength is 348nm, and theoretical cam curve is calculated by luteolin-7-O-β-D-glucuronide peak should be not less than 5000;
B) the preparation precision of reference substance solution takes by weighing at 105 ℃ of luteolin-7-O-beta d glucopyranosiduronic acid glycosides reference substances that are dried to constant weight in right amount, adds 60% ethanol and makes the solution that every 1ml contains 0.25mg;
C) the preparation precision of need testing solution takes by weighing solids 60mg, puts in the 10ml measuring bottle, with water dissolution and be diluted to scale, shakes up, promptly;
D) accurate respectively reference substance solution and each the 5 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, and obtain the content of luteolin in this product-7-O-beta d glucopyranosiduronic acid glycosides.
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