CN102058641B - Detection method of angelica dahurica, extract and pharmaceutical composition containing angelica dahurica extract - Google Patents
Detection method of angelica dahurica, extract and pharmaceutical composition containing angelica dahurica extract Download PDFInfo
- Publication number
- CN102058641B CN102058641B CN 201010622736 CN201010622736A CN102058641B CN 102058641 B CN102058641 B CN 102058641B CN 201010622736 CN201010622736 CN 201010622736 CN 201010622736 A CN201010622736 A CN 201010622736A CN 102058641 B CN102058641 B CN 102058641B
- Authority
- CN
- China
- Prior art keywords
- extract
- imperatorin
- oxypeucedanin
- angelica
- angelica dahurica
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Medicines Containing Plant Substances (AREA)
Abstract
The invention provides an angelica dahurica extract including oxypeucedanin of 9.50-14.24mg/g. The invention further provides a medicine composite which is a medicine prepared from angelica dahurica extract as an active component and pharmaceutically acceptable auxiliary materials or auxiliary components. The invention further provides a method for detecting the quality of angelica dahurica crude drug, the angelica dahurica extract or the medicine composite including the angelica dahurica extract by taking the oxypeucedanin as an index component. The contents of the oxypeucedanin and imperatorin are controlled, the qualities of the angelica dahurica crude drug and the angelica dahurica extract are detected, and the quality detection method can be applied to monitoring the quality of the crude drugs, and detecting and distinguishing whether the angelica dahurica is fumigated by sulfur or not; therefore, the invention can effectively monitor the quality of the angelica dahurica medicine; in addition, the medicine has clear effect and stronger controllability.
Description
Technical field
The present invention relates to a kind of Angelica Dahurica extract and quality determining method, belong to drug world.
Background technology
Root of Dahurain angelica beginning is stated from Shennong's Herbal, for commonly used induce sweat, anodyne.The root of Dahurain angelica is the main flow kind of root of Dahurain angelica commodity medicinal material, accounts for 70% of national commodity root of Dahurain angelica output.At present report utilizes the document of the root of Dahurain angelica more, like application number: 200710111315.3, denomination of invention: Whole coumarins extract from root of dahuriae angelica and preparation method thereof, this disclosure of the Invention a kind of total coumarin extract of obtaining and preparation method thereof that from the Chinese medicine root of Dahurain angelica, extracts.The selectivity basis that this extract comprises is Imperatorin, Isomperatorin, 5-hydroxyl-8-methoxyl psoralen, bergapten, or its any or several combination.This extract also comprises any in the compositions such as root of Dahurain angelica element, aqua oxidation peucedanin, different oxypeucedanin, 5-methoxyl-8-hydroxyl psoralen, xanthotoxol or several combination.This extract can be by any one methods such as solvent extraction method, solvent extraction, macroporous absorbent resin method, lead salt precipitation, column chromatography, liquid-liquid counter partographies, or the combination in any of these methods prepares.The summation of various Coumarins composition percentage compositions is 5～100% (w/w) in the prepared Whole coumarins extract from root of dahuriae angelica, and wherein the content of Imperatorin, Isomperatorin, three kinds of compositions of bergapten accounts for 5～100% (w/w) of whole general coumarin content.
The habitat processing method of the root of Dahurain angelica is bigger to root of Dahurain angelica quality influence; The main at present sulphur fumigation that adopts, still, its cumarin and volatile oil composition reduce greatly behind a large amount of experiment confirm sulphurings; The drug effect of the root of Dahurain angelica is unstable, and it is most important therefore to control the angelica root quality.Report about root of Dahurain angelica method of quality control has: " Chinese pharmacopoeia version in 2000 is only limited to former plant variety, medicinal material proterties and simple physicochemical identification to the quality control of angelica root, lacks an effective Control of Internal Quality method of cover and a quantizating index.Gao Ying, etc., the comparison of Imperatorin content assaying method in the root of Dahurain angelica; Chengdu University of Traditional Chinese Medicine's journal; The 29th the 4th phase of volume of the I2 month in 2006, another kind of Imperatorin content assaying method is disclosed, wherein; With methyl alcohol: water (70: 30) is moving phase, flow velocity 1ml/min, 25 ℃ of column temperatures, detects wavelength 300nm, and number of theoretical plate calculates by the Imperatorin peak and is not less than 3000.This method can be measured the content of three kinds of main Coumarins compositions simultaneously.Zhong Shihong, etc., the research of root of Dahurain angelica HPLC finger-print, World Science technology-traditional Chinese medicine modernization standard and standard, 2005, the seven the 6th phases of volume.Sample is carried out HPLC measure, adopt several different methods such as principal component analysis (PCA), cluster analysis and similarity evaluation to estimate, and different tests project sample in the root of Dahurain angelica GAP has been carried out the comparison of finger-print.The result: sulphuring and not the finger-print of sulphuring sample exist than big-difference, set up the sulphuring and the finger-print of sulphuring angelica root not respectively.At present report quantitatively above-mentioned or qualitatively method can't effectively control the quality of the root of Dahurain angelica.
Summary of the invention
Technical scheme of the present invention has provided a kind of Angelica Dahurica extract, and another technical scheme of the present invention has provided the quality determining method of angelica root and Angelica Dahurica extract.
The invention provides a kind of Angelica Dahurica extract, it contains oxypeucedanin 9.50-14.24mg/g.It also contains Imperatorin 5.52-8.28mg/g.
Angelica Dahurica extract of the present invention is the water or the extractive with organic solvent of angelica root.It is to be prepared from following method: get the angelica root meal, add the 50-95% alcohol extract; Extract promptly gets Angelica Dahurica extract through 60-70 ℃ of concentrating under reduced pressure drying.Wherein, described alcohol extract method is percolation or ultrasonic extraction.
The HPLC finger-print of described Angelica Dahurica extract is as shown in Figure 2, and chromatographic condition is: chromatographic column: Diamonsil
TMC
18HPLC chromatographic column (250 * 4.6mm, 5 μ m); Moving phase: methyl alcohol-tetrahydrofuran-water (56: 9: 35); Flow velocity: 1.0ml/min; Column temperature: 30 ℃; Sample size: 10 μ l; Detect wavelength: 305nm; Number of theoretical plate should be not less than 4000 in oxypeucedanin, Imperatorin.
The root of Dahurain angelica of the present invention or Angelica Dahurica extract; Be meant the root of Dahurain angelica without sulphuring; Wherein, the root of Dahurain angelica is the root of Umbelliferae archangel root of Dahurain angelica Angelica dahurica (Fisch.Ex Hoffm.) or Radix angelicae dahuricae Angelica dahurica (Fisch.Ex Hoffm.) Benth.et Hook.f.var.formosana (Boiss.) Shan et Yuan.
The present invention also provides a kind of pharmaceutical composition, and it is to be active component to contain described Angelica Dahurica extract, adds the medicament that acceptable accessories or complementary composition are prepared from.
Wherein, described medicament is capsule, tablet, granule, pill, oral liquid, soft capsule or pill.
The invention provides a kind of method for quality that detects angelica root, Angelica Dahurica extract or contain the pharmaceutical composition of Angelica Dahurica extract; It is characterized in that: it is that index components is carried out quality control with oxypeucedanin, Imperatorin simultaneously, and it comprises the steps:
A, through high effective liquid chromatography for measuring, the ultraviolet detection wavelength is 305nm;
B, get oxypeucedanin, Imperatorin reference substance, add methyl alcohol and process reference substance solution;
The preparation of c, need testing solution: get angelica root, extract or preparation, add methyl alcohol, power 300W, the ultrasonic Extraction 0.5-1.5 of frequency 50kHz hour, take out, put coldly, be diluted to scale with methyl alcohol, shake up, filter, get subsequent filtrate, promptly get;
D, accurate reference substance solution and the need testing solution injection liquid chromatograph drawn of difference are measured, and promptly get.
Wherein, the described chromatographic condition of a step is: chromatographic column: Diamonsil
TMC
18HPLC chromatographic column (250 * 4.6mm, 5 μ m); Moving phase: methyl alcohol-tetrahydrofuran-water (56: 9: 35); Flow velocity: 1.0ml/min; Column temperature: 30 ℃; Sample size: 10 μ l; Detect wavelength: 305nm; Number of theoretical plate should be not less than 4000 in oxypeucedanin, Imperatorin.
Contain oxypeucedanin 0.12-0.23%w/w, Imperatorin 0.11-0.22%w/w in the described angelica root.
The present invention is through detect the content of oxypeucedanin, Imperatorin simultaneously; Can effectively control the quality of angelica root and extract; The method of quality control of angelica root of the present invention can be used for monitoring the quality of medicinal material, is used for detecting, differentiates the sulphuring and the sulphuring root of Dahurain angelica not, can monitor the quality of root of Dahurain angelica medicine effectively; The drug effect of medicine of the present invention is clear and definite, and controllability is stronger.
Description of drawings
Fig. 1 root of Dahurain angelica extraction process of the present invention process flow diagram
(moving phase is methyl alcohol to Fig. 2 need testing solution chromatogram: water 65: 35)
Fig. 3 reference substance chromatogram (6.00min is that oxypeucedanin, 8.42min are Imperatorin)
Fig. 4 need testing solution chromatogram (bulk drug is sulphuring not, lot number 091001)
Fig. 5 test sample chromatogram (bulk drug sulphuring, lot number 091011)
Fig. 6 oxypeucedanin canonical plotting
Fig. 7 Imperatorin canonical plotting
Fig. 8 oxypeucedanin reference substance chromatogram
Fig. 9 oxypeucedanin reference substance absorption curve
Figure 10 Imperatorin reference substance absorption curve
Figure 11 need testing solution chromatogram (moving phase is methanol-water 70: 30)
Figure 12 need testing solution chromatogram (the not sulphuring root of Dahurain angelica)
Figure 13 need testing solution chromatogram (the sulphuring root of Dahurain angelica)
Figure 14 Imperatorin reference substance chromatogram
Figure 15 need testing solution 60min chromatogram
Embodiment
The preparation method of embodiment 1 Angelica Dahurica extract
Angelica root breaks into meal, with the speed diacolation of 2.5ml/minkg, collects the soup of 6 times of amounts behind the 50% alcohol solution dipping 24h, under 60～70 ℃ temperature, carries out the concentrating under reduced pressure moulding, and drying gets Angelica Dahurica extract of the present invention.
The preparation of embodiment 2 Angelica Dahurica extracts of the present invention
The angelica root meal adds 70% ethanol of 12 times of amounts, power 80W, and ultrasonic Extraction 50min under 55 ℃ of temperature extracts 3 times, and method for concentration is drying to obtain Angelica Dahurica extract of the present invention with embodiment 1.
The preparation of embodiment 3 Angelica Dahurica extracts of the present invention
Get angelica root, extract, concentrate by embodiment 1 method, the soup of gained carries out spray drying.
Spray-dired condition is: the feed liquor temperature of charge: 70～80 ℃; Medicinal extract relative density: 1.08～1.10 (60 ℃); Feed liquor speed: 60～70ml/min; EAT: 138～140 ℃; Leaving air temp: 83～85 ℃.Get Angelica Dahurica extract.
The preparation of embodiment 4 medicines of the present invention
Method preparation according to embodiment 3 when carrying out spray drying, adds the dextrin of 40% dried cream weight, gets granule.
The preparation of embodiment 5 medicines of the present invention
Get the Angelica Dahurica extract that embodiment 1 prepares, add acceptable accessories, prepare tablet.
The preparation of embodiment 6 medicines of the present invention
Get the soup that embodiment 1 concentrating under reduced pressure obtains, after the removal of impurities, add acceptable accessories, prepare oral liquid.
Embodiment 1-6 is to the preparation method of Angelica Dahurica extract of the present invention and medicine; Should not be construed as is the restriction to protection domain of the present invention; All based on above-mentioned technological thought, the modification, replacement, the change that utilize ordinary skill knowledge and customary means to make all belong to scope of the present invention.
Embodiment 7 Angelica Dahurica extract extraction process parameter shaker tests of the present invention
One, set up index investigation method
The Coumarins composition is its main active in the root of Dahurain angelica, multiple pharmacological effect such as modern pharmacology experiment shows that furocoumarin contained in the root of Dahurain angelica has antipyretic-antalgic, anti-inflammatory, relievings asthma, step-down, antibiotic, spasmolysis, photosensitive, activation sympathetic system hormone.In addition, Imperatorin is one of its main effective constituent.So confirm with general coumarin, Imperatorin to be chemical index.For taking into account moulding, with the reference index of dried cream rate as technical study.
1. the mensuration of dried cream rate
Precision pipettes each extract 25ml respectively, put in the evaporating dish that is dried to constant weight, and behind evaporate to dryness in the water-bath, residue 105 ℃ of dryings 3 hours in baking oven, cooling is 30 minutes in the dislocation exsiccator, and accurate the title, decided weight, calculates dried cream rate (%).
2. general coumarin Determination on content
With the Imperatorin is reference substance, adopts AAS to measure at the 300nm place.Typical curve is: A=48.998C-0.0012, (R
2=0.9999, n=6) good in 5.15～18.025 μ g/ml scope internal linear relation; Method precision: RSD=0.16%; Method stability: the sample test liquid is stable in 8h, RSD=2.19%.The method recovery is 98.25%, RSD=1.70%.
3. Imperatorin Determination on content
Adopt high effective liquid chromatography for measuring.Chromatographic condition: use octadecyl silane to be filling agent; Methyl alcohol: water (70: 30) is moving phase; The detection wavelength is 300nm.Column temperature: 30 ℃, flow velocity is 1.0ml/min, sample size: 10 μ L.
Typical curve is: Y=7E+06X+21286, R=0.9999 is good in 103～927 μ g scope internal linear relation; Method precision: RSD=2.12%; Method stability: sample supplies examination solution stable in 120h, RSD=2.35%; The method recovery is 98.68%, RSD=1.96%.
Two, Angelica Dahurica extract Study on Preparation of the present invention
1. extraction process is investigated
In the preliminary experiment root of Dahurain angelica percolation extraction process has been carried out the orthonormal design of experiments investigation; The result shows not statistically significant; Analyzing reason is owing to cause like the elasticity of fitted tube, the addition of fresh liquor, the uncertain factors such as thickness of cotton in the fitted tube process, finally selects single factor to investigate.
(1) extraction solvent is investigated
Take by weighing two parts of 100g root of Dahurain angelica meal, water and 95% ethanol extract respectively, and extract concentrates and is settled to certain volume, press the content of general coumarin and Imperatorin in content assaying method calculating water extract and the alcohol extract.The investigation result of table 1 extraction solvent
(2) investigation of medicinal material granularity
Take by weighing each 3 parts of the meal of angelica root, meal, middle powder, every part of 100g, the ethanol swelling with 50% is after 1 hour; Fitted tube flooded after 4 hours, with the speed diacolation extraction of 0.25ml/min; Collect percolate 600ml sampling, measure cumarin, Imperatorin, dried cream rate respectively, the result sees table 2:
The investigation result of table 2 medicinal material granularity
The result shows: medicinal material is best with the effect of meal diacolation, and medicinal powder is thick excessively, and effective constituent is not easy to leach, and pulverizing medicinal materials gets too thin, and soup is difficult for flowing out, blockage phenomenon often in the amplification test.Confirmed that thus with meal be best grinding particle size.
(3) Different Extraction Method is extracted the result relatively
Get 3 parts of root of Dahurain angelica meal, every part of about 100g, it is fixed to claim, and portion soaked 4 hours after 1 hour with 50% ethanol swelling, with the speed diacolation extraction of 0.25ml/min, collected percolate to 600ml; A with 50% alcohol reflux 3 times, 600ml at every turn, 1 hour; Another part is with 6 times of amount 50% ethanol room temperature ultrasonic Extraction 30min, and 2 times, each collects 6 times of medicine amount liquids.
The investigation result of table 3 method for distilling
Can be known by table: percolation is superior to other method for distilling to the extraction effect of index components, and so selects percolation to extract.
The comprehensive above result that investigates confirms the process route that extracts with ethanol percolation.
(4) investigation of solvent concentration
Take by weighing 5 parts of the meal of angelica root, every part of 100g, respectively with the Different concentrations of alcohol swelling after 1 hour, fitted tube flooded after 4 hours, with the speed diacolation of 0.25ml/min, collected percolate to 600ml.The investigation result of table 4 solvent concentration
The result shows: 50%, 65%, 75% ethanol is suitable to the extraction effect of general coumarin, and dried cream rate reduces successively, and still along with the reduction of determining alcohol, the impurity of extraction is just many more.Later stage in conjunction with cost consideration, is fixed tentatively 50% ethanol percolation through the comparison to 50% and 70% ethanol.
(5) investigation of diacolation speed
Take by weighing 4 parts of the meal of angelica root, every part of 100g, the ethanol swelling with 50% is after 1 hour; Fitted tube floods after 4 hours, respectively with 0.25ml/min; 0.5ml/min; 1.0ml/min the speed diacolation of 2.0ml/min extracts (in view of the time-saving consideration of pilot scale amplification test), collects percolate 600ml.
The investigation result of table 5 diacolation speed
The result shows: the diacolation VELOCITY EXTRACTION effect of 0.25ml/min is superior to other infiltration rate.So confirm that the diacolation speed of 100g medicinal material is 0.25ml/min, promptly diacolation speed is 2.5ml/minkg.
(6) investigation of soak time
Take by weighing 4 parts of the meal of angelica root, every part of 100g, the ethanol swelling with 50% is after 1 hour, fitted tube, soak 4h, 8h, 12h, 24h respectively after, extract with the speed diacolation of 0.25ml/min, collect percolate 600ml.
The investigation result of table 6 soak time
The result shows: it is best to soak the 24h extraction effect, confirms that soak time is 24h.
(7) investigation of collecting amount
Take by weighing the meal 100g of angelica root, the ethanol swelling with 50% is after 1 hour, fitted tube, soak 24h after, extract with the speed diacolation of 0.25ml/min, be to collect unit Fractional Collections percolate with 100ml, collect 10 parts altogether, promptly total collection is 1000ml.
The investigation result of table 7 collecting amount
The result shows: proper when the percolate collecting amount is 600ml, extract fully basically, and confirm that collecting amount is 600ml.Consider to amplify and produce,,, when guaranteeing that most effective constituents are oozed out, also shortened the production cycle like this, practiced thrift a large amount of ethanol the solvent that goes out of the 5th, six part of percolate as this medicine of next group so collect preceding four parts percolate.
(8) confirmatory experiment
According to the result that above experiment of single factor is investigated, confirmed optimum extraction process: angelica root breaks into meal, with the speed diacolation of 2.5ml/minkg, collects the soup of 6 times of amounts behind the 50% alcohol solution dipping 24h.Take by weighing 3 parts of angelica roots, every part of 100g experimentizes by set process conditions, measures general coumarin, Imperatorin content and the dried cream rate of above-mentioned sample liquid respectively, and the result sees as follows:
Table 8 confirmatory experiment result
The confirmatory experiment result shows that this technology has repeatability, stablizes feasible.
In addition, we investigate also right ultrasonic extraction process.
Method: the extracted amount with general coumarin is index (combining dried cream rate), on the experiment of single factor basis, adopts orthogonal design that technology is optimized, and has investigated the influence of factors such as temperature, solid-liquid ratio, power, extraction time to general coumarin.
Result: draw optimised process: medicinal material coarse powder adds 70% ethanol of 12 times of amounts, and power 80W extracts 50min under 55 ℃ of temperature, extracts 3 times.
2. impurity removal process research
By extraction process gained percolate paste-forming rate is 14%～17%, and moulding easily adds the consideration of producing to amplification, so decision wouldn't be selected the chemical subtraction method for use, we have carried out the investigation of centrifugal removal of impurities to percolate for this reason.
Get two parts of the percolates that extract by set technology, a directly centrifugal with soup; Another part soup is evaporated to every ml soup and is equivalent to centrifugal (5000r/min, centrifugal 1h) behind the 0.5g crude drug.The result is following:
Table 9 centrifugal method is investigated the result
The centrifugal method result
Soup is centrifugal poor effect directly, and soup is not seen layering
Soup concentrates the centrifugal soup layering in back, and a small amount of canescence, oily mater are arranged at the centrifuge tube bottom
To percolate before centrifugal, concentrate the mensuration that supernatant and centrifugal gained deposition after centrifugal are carried out Imperatorin and dried cream rate, the result is following:
The centrifugal impurity removal process of table 10 is investigated the result
This shows that the dried cream rate in the centrifugal back of percolate reduces not remarkable.
Percolate is in the concentrating under reduced pressure process, and ethanol is recovered, and solvent is mainly water in the concentrate, and Imperatorin is a liposoluble constituent, and so dissolving hardly in the WS is the deposition of formation.
So confirm percolate is directly concentrated moulding.
3. concentration technology research
The angelica dahurica coumarin constituents is met thermally labile, and is relatively comprehensive, selects the method for concentrating under reduced pressure.Be necessary that at first the thermal stability of extract is investigated when concentrating, method is following: experimental technique and result: get 3 parts of extracts, every part of 200ml is respectively at placing 2 hours in 80 ℃ of water-baths; 4 hours, 8 hours, put cold; Supply volume, assay is carried out in sampling, and the result is following:
Table 11 root of Dahurain angelica thermal stability is investigated the result
Disposal route Imperatorin (mg/g)
Be untreated 1.3716
80 ℃ 2 hours 1.3709
80 ℃ 4 hours 1.3698
80 ℃ 8 hours 1.3240
The result shows, the root of Dahurain angelica 80 ℃ following 4 hours with interior more stable, heated time is long, causes effective constituent to be decomposed easily, content reduces.Because concentrating under reduced pressure efficient is high, keep fluid temperature in the time of 60～70 ℃, concentration time all is no more than 2 hours.On the other hand, temperature is low excessively, reduces thickening efficiency, the too high active constituent content that then influences of temperature.So confirm that these article should carry out concentrating under reduced pressure under 60～70 ℃ temperature.
4. drying process research
Because it is fast that spray drying has a rate of drying than additive method, the material heated time is short, the thermal sensitivity composition is destroyed few; Production process is simple, and is easy and simple to handle, is easy to control of quality; Be applicable to industrial continuous big production, it is few that institute adds auxiliary material, thereby reduce dose; Reach characteristics such as the exquisite effect of Chinese medicine, therefore adopt spray-dired method that material is carried out drying.
Influencing spray-dired principal element has: the kind of medicinal extract relative density, auxiliary material and consumption, feed liquor temperature of charge, charging rate, EAT, leaving air temp etc.
(1) investigation of different auxiliary material
Consider cost and adaptability, dextrin, soluble starch, three kinds of auxiliary materials of lactose are investigated.Extract after medicinal material concentrated is divided into 4 equal portions; Press 50% of dry extract respectively and add 3 kinds of auxiliary materials, make dissolving or be uniformly dispersed; 1 part does not add any auxiliary material in addition, spray-dried separately behind the abundant mixing, and the result is following:
The spray-dired comparison of table 12 different auxiliary material
This shows that the powder that spray drying obtains behind the adding dextrin is loose, sticking hardly wall, color and luster is better.Soluble starch is insoluble to medicinal extract after adding, and has deposition to produce; Lactose high temperature is prone to softening, and material is flow-like and adheres to inwall when dry.So selecting dextrin is its spray-dired auxiliary material.
(2) investigation of auxiliary material dextrin consumption:
According to these article Study on extraction process, confirmed with 50% ethanolic solution as extraction solvent.The medicinal extract spray drying of alcohol extracting is difficult, and because of its stickiness is big, in spray-dired process, have the auxiliary material that resists glutinous and peptizaiton normal the adding, to improve the character of material.Combine these article situation in the experiment, the dextrin addition is investigated during to these article drying, and the result is following.
The investigation of table 13 supplementary product consumption
Dextrin addition/dried cream amount is glued the wall degree
0% sticking wall is serious
20% sticking wall is more serious
40% not sticking wall, powder is loose
60% not sticking wall, powder is loose, and color and luster is even
Can know that by the result when the dextrin consumption is 40% when above of dried cream amount in the medicinal extract, drying effect is better, can avoid sticking wall, obtains loose powder.The later stage test shows the dextrin that adds dried cream amount 30%, and wall sticking phenomenon still exists, and is dried cream amount 40% so add the amount of dextrin when confirming spray drying.
(3) investigation of medicinal extract relative density
The medicinal extract (dextrin that contains dried cream amount 50%) that relative density is respectively 1.04,1.08,1.10,1.12 (60 ℃) carries out spray drying (EAT: 138～140 ℃, leaving air temp: 82～85 ℃), observes the dry materials situation, and the result is following:
The screening of table 14 medicinal extract relative density
Medicinal extract relative density (60 ℃) dry materials situation
1.04 not sticking wall, powder is loose
1.08 not sticking wall, powder is loose
1.10 not sticking wall, powder is loose
1.12 slightly sticking wall, color is dark
The result shows: the medicinal extract relative density is chosen in 1.08～1.10 (60 ℃) can get quality powdered extract powder preferably when carrying out spray drying.The medicinal extract relative density is too high, and the medicinal extract viscosity is big, is prone to blocking pipe, the atomizing difficulty, and the particle that atomizing forms is big, and material is prone to cohere, the discharging color burn, even the burnt phenomenon of sticking with paste appears; Otherwise relative density is low excessively, needs the also corresponding increase of the water yield of evaporation, thereby prolongs the production time, reduces production efficiency.So confirm that the medicinal extract relative density is 1.08～1.10 (60 ℃).
(4) investigation of EAT, leaving air temp
To contain medicinal extract (1.10,60 ℃ of the relative densities) charging of the dextrin of dried cream amount 50%, under different EATs, leaving air temp, carry out spray drying respectively, observe the dry materials situation.The result is following.
The investigation of table 15 EAT, leaving air temp
Can know that by above-mentioned experimental result EAT is 133～135 ℃, glues wall when leaving air temp is 80～82 ℃ on a small quantity, a small amount of block is arranged; EAT is 138～140 ℃, when leaving air temp is 83～85 ℃, effect is better, obtains yellow loose powdered; EAT is 143～145 ℃, when leaving air temp is 86～88 ℃, color burn is even burnt sticking phenomenon occurs.Be respectively 138～140 ℃, 83～85 ℃ so confirm EAT, leaving air temp.
(5) confirming of charging fluid temperature
Material need keep uniform temperature in the past getting into the spray tower drying, helped reducing the viscosity of material, made the moisture back evaporation rapidly in getting into drying tower in the material, thereby shortened drying time, improved drying efficiency.
Investigating result's (investigate early stage) by root of Dahurain angelica thermal stability in these article concentration technology can know, these article 80 ℃ 4 hours with interior more stable.For taking into account drying efficiency and index components, confirm that the material holding temperature is 70～80 ℃.
(6) investigation of charging rate
Charging rate also directly affects drying process, and excessive velocities has surpassed the atomizing ability of atomizer, be prone to materialization not thoroughly, wall sticking phenomenon; Charging rate is slow excessively, prolongs the production time, reduces production efficiency.The investigation result is following:
The investigation of table 16 charging rate
Charging rate (ml/min) dry materials situation
50～60 dried powders
60～70 dried powders
70～80 materials bonding
The result shows: charging rate is controlled in 50～60ml/min scope, all can obtain dried powder, for taking into account drying efficiency, confirms that charging rate is 60～70ml/min.Also should do corresponding adjustment in the production according to the intrinsic parameter of equipment.
(7) Imperatorin Determination on content before and after the drying
Carry out spray drying by gained spray drying condition, collect dried powder, measure Imperatorin content, the result is following:
Imperatorin content relatively before and after table 17 spray drying
Sample Imperatorin content (mg/g)
Spray drying preceding 1.4531
After the spray drying 1.4496
The result shows: effective constituent Imperatorin content does not have significant change before and after the spray drying, and definite spray drying condition is rationally feasible.
(8) mensuration of spray-dried powders water cut
By " 2005 editions one appendix IX H of Chinese pharmacopoeia aquametry, first method is measured the water cut of three batches of spray-dried powders, and the result is following.
The mensuration result of table 18 spray-dried powders water cut
The result shows: the dry extract water cut that spray drying obtains is less.
Comprehensive above-mentioned result of study, confirm that the spray drying condition is:
The addition of auxiliary material dextrin: 40% of dried cream amount; Feed liquor temperature of charge: 70～80 ℃; Medicinal extract relative density: 1.08～1.10 (60 ℃); Feed liquor speed: 60～70ml/min; EAT: 138～140 ℃; Leaving air temp: 83～85 ℃.Through demonstration test, the result shows that this technology is rationally feasible.
5. preparation technology
By above result of study, can tentatively draw Angelica Dahurica extract preparation technology, process chart is as shown in Figure 1.
The content of oxypeucedanin, Imperatorin in the medicinal material Angelica Dahurica extract of the different places of production of embodiment 8 high effective liquid chromatography for measuring
1, the selection of chromatographic condition
On the basis of chromatographic condition, as moving phase, the degree of separation of oxypeucedanin is still not good in the sample, sees Fig. 2 with methanol-water (65: 35).After the adjustment moving phase (methyl alcohol-tetrahydrofuran-water 56: 9: 35), each component obtains separating effect (reference substance and sample chromatogram figure see Fig. 3,4,5) preferably.The peak shape symmetry of oxypeucedanin, Imperatorin element, sharp-pointed, theoretical cam curve is 6000.
2, chromatographic condition
Chromatographic column: Diamonsil
TMC
18HPLC chromatographic column (250 * 4.6mm, 5 μ m)
Moving phase: methyl alcohol-tetrahydrofuran-water (56: 9: 35)
Flow velocity: 1.0ml/min
Column temperature: 30 ℃
Sample size: 10 μ l
Detect wavelength: 305nm
3, the preparation of need testing solution
3.1 the investigation of extraction solvent
According to the character of oxypeucedanin, Imperatorin, the while list of references, plan is investigated with methyl alcohol, ethyl acetate, 95% ethanol, is carried out the investigation of extraction solvent.
Get 3 parts of medicines of the present invention (by embodiment 4 preparations, 091002 batch), every part of accurate title of 0.2g is fixed, puts in the 50ml measuring bottle; Add methyl alcohol, 95% ethanol, each 45ml of ethyl acetate respectively, sonicated (power 300W, frequency 50kHz) 1 hour; Filter, residue washs with corresponding solvent, filters.After filtrating water-bath Back stroke is done, add methyl alcohol and be diluted to 50ml, promptly get.With 0.45 μ m filtering with microporous membrane, draw 10 μ l before the sample introduction, inject liquid chromatograph, measure, calculate peak area according to aforementioned chromatographic condition.Inject oxypeucedanin, Imperatorin reference substance solution simultaneously, calculate the content of oxypeucedanin, Imperatorin in each sample solution by external standard method.The method operation repetitive twice like this, and the result sees table 19.
The investigation of table 19 extraction solvent
Above result shows that the methanol extraction effect is better, therefore selects methyl alcohol as extraction solvent.
3.2 the investigation of method for distilling
Relatively ultrasonic Extraction, refluxing extraction, cold soaking extract the influence to the Imperatorin extraction effect.Get 3 parts of medicines of the present invention (091002 batch) 0.2g, the accurate title, decide.Portion is put in the 50ml measuring bottle, adds methyl alcohol 45ml, sonicated (power 300W, frequency 50kHz) 1 hour.Portion is put in the 100ml conical flask, adds methyl alcohol 45ml, leaves standstill 24 hours.Portion is put in the 100ml round-bottomed flask, adds methyl alcohol 45ml, refluxing extraction 1 hour.Each extract filters, and is diluted in the 50ml measuring bottle with methyl alcohol, shakes up, and filters, and gets subsequent filtrate, promptly gets.With 0.45 μ m filtering with microporous membrane, draw 10 μ l before the sample introduction, inject liquid chromatograph, measure the content of Imperatorin in the calculation sample according to aforementioned chromatographic condition.The method operation repetitive is twice like this.The result sees table 20.
Table 20 Different Extraction Method extraction effect relatively
The result shows that the refluxing extraction method is relatively poor, and ultrasonic Extraction and cold soaking extract better, consider that the cold soaking time is oversize, therefore selects ultrasonic extracting method for use.
3.3 the investigation of ultrasonic Extraction time
Get 3 parts of medicines of the present invention (091002 batch), every part of 0.2g, the accurate title, decide, and puts in the 50ml measuring bottle, adds methyl alcohol 45ml; 0.5 hour, 1 hour, 1.5 hours each extracts of sonicated (power 300W, frequency 50kHz) take out, put cold, with methanol constant volume to scale; Shake up, filter, get subsequent filtrate, promptly get.With 0.45 μ m filtering with microporous membrane, draw 10 μ l before the sample introduction, inject liquid chromatograph, measure the content of Imperatorin in the calculation sample according to aforementioned chromatographic condition.The method operation repetitive is twice like this.The result sees table 21.
Extraction effect of different ultrasonic Extraction time of table 21 relatively
The result shows that the Imperatorin extraction effect was good slightly in ultrasonic 1.5 hours, but took all factors into consideration, and selected ultrasonic 1 hour.
Can know that to sum up the need testing solution preparation method is: get the about 0.2g of medicine of the present invention, the accurate title, decide, and puts in the 50ml measuring bottle, adds methyl alcohol 45ml; Sonicated (power 300W, frequency 50kHz) 1 hour is taken out, and puts coldly, is diluted to scale with methyl alcohol; Shake up, filter, get subsequent filtrate, promptly get.
4, the investigation of linear relationship
Precision takes by weighing oxypeucedanin reference substance 4.70mg, puts in the 100ml measuring bottle, adds dissolve with methanol and is diluted to scale, shakes up, and promptly gets the oxypeucedanin reference substance solution of 47.0 μ g/ml.Draw reference substance solution 4 μ l, 8 μ l, 12 μ l, 16 μ l, 20 μ l respectively, carry out stratographic analysis, measure peak area by above-mentioned chromatographic condition.The result sees table 22.With the peak area is ordinate, and the amount of oxypeucedanin is a horizontal ordinate, and the drawing standard curve gets regression equation, sees Fig. 6:
y＝1458430x-12190 R
2＝0.999
Precision takes by weighing Imperatorin reference substance 4.82mg, puts in the 100ml measuring bottle, adds dissolve with methanol and is diluted to scale, shakes up, and promptly gets the Imperatorin reference substance solution of 48.2 μ g/ml.Draw reference substance solution 4 μ l, 8 μ l, 12 μ l, 16 μ l, 20 μ l respectively, carry out stratographic analysis, measure peak area by above-mentioned chromatographic condition.The result sees table 23.With the peak area is ordinate, and the amount of Imperatorin is a horizontal ordinate, and the drawing standard curve gets regression equation, sees Fig. 7:
y＝3026918x+5372 R
2＝0.999
Table 22 linear relationship is investigated the result
Above result shows, shows the sample feeding amount in 0.188～0.940 μ g, and oxypeucedanin peak area value and sample size present good linear relationship; The sample feeding amount is in 0.1928～0.9640 μ g, and Imperatorin peak area value and sample size present good linear relationship.
5, precision test
Accurate respectively oxypeucedanin reference substance test solution (47.0 μ g/ml) and each 10 μ l of Imperatorin reference substance test solution (48.2 μ g/ml) of drawing, continuous sample introduction 5 times is measured peak area value, and the result sees table 23.
Table 23 precision is investigated the result
Mensuration result shows: this method precision is good.
6, stability test
6.1 the stability of reference substance solution
Accurate oxypeucedanin reference substance test solution (47.0 μ g/ml) and each 10 μ l of Imperatorin reference substance test solution (48.2 μ g/ml) of drawing respectively 0,2, measured once in 4,8,12,24 hours, measured the result and saw table 24.
Mensuration result shows: reference substance solution is good at 24 hours internal stabilities of placement.
The stability test of table 24 reference substance solution
6.2 the stability of need testing solution
Get medicine 0.2g of the present invention, the accurate title, decide, and handles by sample preparation methods, promptly gets need testing solution.Press the sample determination method, measured once at 0,2,4,8,12,24 hour respectively.The result sees table 25.
The stability test of table 25 need testing solution
Mensuration result shows: need testing solution place in 24 hours basicly stable.
7, reappearance test
Get parallel 5 parts of medicine of the present invention, every part of 0.2g, the accurate title, decide, and handles by sample preparation methods, promptly gets need testing solution.Press the sample determination method, record the sample peak area, calculate the content of Imperatorin, the result sees table 26.
The test of table 26 reappearance
Mensuration result shows: oxypeucedanin, Imperatorin element contain heavily in this method working sample, and reappearance is good.
8, application of sample recovery test
Adopt the average recovery method, get the about 0.2g of medicine of the present invention of known content respectively, the accurate title, decide; Each accurate an amount of oxypeucedanin, Imperatorin reference substance of adding; Press the operation of sample determination method, record the content of oxypeucedanin in the sample, Imperatorin, calculate recovery rate.The result sees table 27,28.
Table 27 oxypeucedanin recovery experimental result
Table 28 Imperatorin recovery experimental result
Mensuration result shows: this method has the recovery preferably.
9, the mensuration of sample
Get each about 0.2g of test agent in 11 crowdes, accurate claim surely, put in the 50ml measuring bottle, add methyl alcohol 45ml, sonicated (power 300W, frequency 50kHz) 1 hour is taken out, and puts coldly, is diluted to scale with methyl alcohol, shakes up, and filters, and gets subsequent filtrate, promptly gets.Accurate each need testing solution 20 μ l that draw inject liquid chromatograph, measure peak area, calculate the content of Imperatorin in each batch sample.See table 29.
Table 29 assay result (n=2)
Visible by above mensuration result: the oxypeucedanin content in the 10 batches of medicines of the present invention is the highest by 0.88%, and minimum 0.80%, average 0.84%; Imperatorin content is the highest by 0.47%, and is minimum 0.50%, average 0.49%, considers differences such as quality of medicinal material, so stipulate oxidation of drug peucedanin (C of the present invention
16H
14O
5) must not be less than 0.60%, Imperatorin (C
16H
14O
4) must not be less than 0.40%.
The auxiliary material amount is 40% of a dried cream amount in known the invention described above medicine; Just auxiliary material accounts for 29% of total extract, and dried cream accounts for 71% of total extract, therefore; Oxypeucedanin in the Angelica Dahurica extract, the content of Imperatorin can be converted by last table result and obtain, and the result sees table 30.
After the conversion, before and after the oxidation of 10 batches of Angelica Dahurica extracts cellulose content between 11.3-12.5mg/g, average 11.8mg/g; Imperatorin content is considered differences such as quality of medicinal material between 6.5-7.3mg/g, so regulation Angelica Dahurica extract oxypeucedanin (C
16H
14O
5) content is between 9.50-14.24mg/g, Imperatorin (C
16H
14O
4) content is between 5.52-8.28mg/g.
According to above-mentioned condition, we have measured bulk drug simultaneously is the middle test agent (091011 batch) of sulphuring, and its Imperatorin content is 3.79mg/g, and the content of oxypeucedanin is lower than the range of linearity.It is thus clear that, be that oxypeucedanin content can't be measured in the prepared extract of raw material with the sulphuring root of Dahurain angelica, the content of Imperatorin has reduced about 47%.
The quality determining method of embodiment 9 angelica roots of the present invention
1, instrument and reagent
Waters2695 type high performance liquid chromatograph
PDAD Waters 2996 types
Electronic analytical balance BP121S (Beijing Sartorius balance company limited)
Oxypeucedanin reference substance (the HPLC area normalization method is confirmed for self-control, purity 99.5%)
The Imperatorin reference substance is available from Nat'l Pharmaceutical & Biological Products Control Institute, and lot number 110826-200712 methyl alcohol, tetrahydrofuran are chromatographically pure (German Fisher), and water is double distilled water, and it is pure that all the other reagent are analysis.
2, the purity test of oxypeucedanin
TLC method precision takes by weighing oxypeucedanin 2.1mg, and dissolve with methanol is settled to 10ml.According to " 2005 editions one (appendix VI B) method experiment of Chinese pharmacopoeia; Draw 5 μ l, 10 μ l, 20 μ l points respectively on same silica gel g thin-layer plate; Use sherwood oil-ether (5: 3), chloroform-methanol (100: 2), three kinds of development systems of sherwood oil-acetone (5: 1) to launch respectively; Sulfuric acid-ethanol colour developing all is single spot in the thin-layer chromatography; Experience under ultraviolet lamp (365nm) all is the yellow fluorescence spot.
HPLC method precision takes by weighing oxypeucedanin 1.0mg, and dissolve with methanol is settled to 100ml.Get 10 μ l and inject high performance liquid chromatograph, under 310nm, inspect, not seeing has impurity peaks to occur.See Fig. 8.
3 detect the selection of wavelength
Get the oxypeucedanin reference substance and process the solution that contains oxypeucedanin 10.7 μ g/ml, get the Imperatorin reference substance and process with dissolve with methanol and contain Imperatorin 11.6 μ g/ml with dissolve with methanol.Other gets blank methanol solution, on UV-1100 type spectrophotometer, draws ultra-violet absorption spectrum in the scanning of 200～400nm wavelength coverage.Imperatorin has an absorption peak at 301nm wavelength around place, and oxypeucedanin has an absorption peak at 310nm wavelength around place, and peak shape changes mild, is convenient to quantitative measurment, finally selects 305nm as detecting wavelength.The result sees Fig. 9,10.
4, the selection of chromatographic condition
Adopt that " the Chinese pharmacopoeia version root of Dahurain angelica in 2005 the item chromatographic condition of regulation is down investigated, and finds that with methanol-water (55: 45) be moving phase when carrying out wash-out, and the retention time of Imperatorin is 56min, and 120min still has chromatographic peak to occur later on.Be adjusted into methanol-water (70: 30) with moving phase early stage; And after column temperature is increased to 30 ℃; The retention time of Imperatorin is 12min in advance, and Imperatorin obtains separating effect (seeing Figure 11) preferably in the sample of investigation back, but the degree of separation of oxypeucedanin does not reach requirement with this understanding.After moving phase was adjusted into methyl alcohol-tetrahydrofuran-water (56: 9: 35), the oxypeucedanin degree of separation was good, and the retention time of Imperatorin is 8min (seeing Figure 12,13) in advance.The peak shape symmetry of oxypeucedanin, Imperatorin, sharp-pointed, theoretical cam curve is 6000.The performance of considering different instruments, chromatographic column possibly there are differences, and should be not less than 4000 (Figure 14) so decide number of theoretical plate in oxypeucedanin, Imperatorin.1 hour chromatogram of need testing solution is seen Figure 15.
5 chromatographic conditions
Chromatographic column: Diamonsil
TMC
18HPLC chromatographic column (250 * 4.6mm, 5 μ m)
Moving phase: methyl alcohol-tetrahydrofuran-water (56: 9: 35)
Flow velocity: 1.0ml/min
Column temperature: 30 ℃
Sample size: 10 μ l
Detect wavelength: 305nm
The preparation of 6 need testing solutions
6.1 the investigation of extraction solvent
According to the character of oxypeucedanin, Imperatorin, the while list of references, plan is investigated with methyl alcohol, ethyl acetate, 95% ethanol, is carried out the investigation of extraction solvent.
Get 3 parts of angelica roots, every part of 0.4g, the accurate title, decide, and puts in the 50ml measuring bottle, adds methyl alcohol, 95% ethanol, each 45ml of ethyl acetate respectively, and sonicated (power 300W, frequency 50kHz) 1 hour filters, and residue washs with corresponding solvent, filters.After filtrating water-bath Back stroke is done, add methyl alcohol and be diluted to 50ml, promptly get.With 0.45 μ m filtering with microporous membrane, draw 10 μ l before the sample introduction, inject liquid chromatograph, measure, calculate peak area according to aforementioned chromatographic condition.Inject oxypeucedanin, Imperatorin reference substance solution simultaneously, calculate the content of oxypeucedanin, Imperatorin in each sample solution by external standard method.The method operation repetitive twice like this, and the result sees table 31.
The investigation of table 31 extraction solvent (n=2)
Above result shows that the methanol extraction effect is better, therefore selects methyl alcohol as extraction solvent.
6.2 the investigation of method for distilling
Relatively ultrasonic Extraction, refluxing extraction, cold soaking extract the influence to the Imperatorin extraction effect.
Get 3 parts of angelica roots, every part of 0.4g, the accurate title, decide.Portion is put in the 50ml volumetric flask, adds methyl alcohol 45ml, sonicated (power 300W, frequency 50kHz) 1 hour.Portion is put in the 50ml conical flask, adds methyl alcohol 45ml, leaves standstill 24 hours.Portion is put in the 50ml round-bottomed flask, adds methyl alcohol 45ml, refluxing extraction 1 hour.Each extract filters, and is diluted in the 50ml measuring bottle with methyl alcohol, shakes up, and filters, and gets subsequent filtrate, promptly gets.With 0.45 μ m filtering with microporous membrane, draw 10 μ l before the sample introduction, inject liquid chromatograph, measure the content of Imperatorin in the calculation sample according to aforementioned chromatographic condition.The method operation repetitive is twice like this.The result sees table 32.
Table 32 Different Extraction Method extraction effect is (n=2) relatively
The result shows that ultrasonic extracting method is good than other two kinds of methods, therefore selects ultrasonic extracting method for use.
6.3 the investigation of ultrasonic Extraction time
Get 3 parts of angelica roots, every part of 0.4g, the accurate title, decide, and puts in the 50ml measuring bottle, adds methyl alcohol 45ml; 0.5 hour, 1 hour, 1.5 hours each extracts of sonicated (power 300W, frequency 50kHz) take out, put cold, with methanol constant volume to scale; Shake up, filter, get subsequent filtrate, promptly get.With 0.45 μ m filtering with microporous membrane, draw 10 μ l before the sample introduction, inject liquid chromatograph, measure the content of Imperatorin in the calculation sample according to aforementioned chromatographic condition.The method operation repetitive is twice like this.The result sees table 33.
Extraction effect of different ultrasonic Extraction time of table 33 is (n=2) relatively
The result shows that the oxypeucedanin extraction effect was good slightly in ultrasonic 1.5 hours, but took all factors into consideration, and still selected ultrasonic 1 hour.
Can know that to sum up the need testing solution preparation method is: get the about 0.4g of angelica root, the accurate title, decide, and puts in the 50ml measuring bottle, adds methyl alcohol 45ml; Sonicated (power 300W, frequency 50kHz) 1 hour is taken out, and puts coldly, is diluted to scale with methyl alcohol; Shake up, filter, get subsequent filtrate, promptly get.
The investigation of 7 linear relationships
Precision takes by weighing oxypeucedanin reference substance 1.07mg, puts in the 100ml measuring bottle, adds dissolve with methanol and is diluted to scale, shakes up, and promptly gets the oxypeucedanin reference substance solution of 10.7 μ g/ml.Draw reference substance solution 2 μ l, 4 μ l, 8 μ l, 12 μ l, 16 μ l, 20 μ l respectively, carry out stratographic analysis, measure peak area by above-mentioned chromatographic condition.The result sees table 34.With the peak area is ordinate, and the amount of oxypeucedanin is a horizontal ordinate, and the drawing standard curve gets regression equation.
Precision takes by weighing Imperatorin reference substance 1.16mg, puts in the 100ml measuring bottle, adds dissolve with methanol and is diluted to scale, shakes up, and promptly gets the Imperatorin reference substance solution of 11.6 μ g/ml.Draw reference substance solution 2 μ l, 4 μ l, 8 μ l, 12 μ l, 16 μ l, 20 μ l respectively, carry out stratographic analysis, measure peak area by above-mentioned chromatographic condition.The result sees table 34.With the peak area is ordinate, and the amount of Imperatorin is a horizontal ordinate, and the drawing standard curve gets regression equation.
Table 34 linear relationship examination result
Above result shows, shows the sample feeding amount in 0.0214～0.214 μ g, and oxypeucedanin peak area value and sample size present good linear relationship; The sample feeding amount is in 0.0232～0.232 μ g, and Imperatorin peak area value and sample size present good linear relationship.
The test of 8 precision
Accurate respectively oxypeucedanin reference substance test solution (10.7 μ g/ml) and each 10 μ l of Imperatorin reference substance test solution (11.6 μ g/ml) of drawing, continuous sample introduction 5 times is measured peak area value, and the result sees table 35.
Mensuration result shows: this method precision is good.
Table 35 precision is investigated the result
9 stability tests
9.1 the stability of reference substance solution
Accurate oxypeucedanin reference substance test solution (10.7 μ g/ml) and each 10 μ l of Imperatorin reference substance test solution (11.6 μ g/ml) of drawing respectively 0,2, measured once in 4,8,12,24 hours, measured the result and saw table 36.
The stability test of table 36 reference substance solution
Mensuration result shows: reference substance solution is good at 24 hours internal stabilities of placement.
9.2 the stability of need testing solution
Get angelica root 0.4g, the accurate title, decide, and handles by sample preparation methods, promptly gets need testing solution.Press the sample determination method, measured once at 0,2,4,8,12,24 hour respectively.The result sees table 37.
The stability test of table 37 need testing solution
Mensuration result shows: need testing solution place in 24 hours basicly stable.
The test of 10 reappearances
Get parallel 5 parts of the root of Dahurain angelica, every part of 0.4g, the accurate title, decide, and handles by sample preparation methods, promptly gets need testing solution.Press the sample determination method, record the sample peak area, the result sees table 38.
The test of table 38 reappearance
Mensuration result shows: oxypeucedanin, Imperatorin cellulose content in this method working sample, reappearance is good.
11 application of sample recovery tests
Adopt the average recovery method, get the about 0.4g of angelica root of known content respectively, the accurate title, decide; Each accurate an amount of oxypeucedanin, Imperatorin reference substance of adding; Press the operation of sample determination method, record the content of oxypeucedanin in the sample, Imperatorin, calculate recovery rate.The result sees table 39,40.
Mensuration result shows: this method has the recovery preferably.
Table 39 oxypeucedanin recovery experimental result
Table 40 Imperatorin recovery experimental result
The mensuration of 12 samples
Get each about 0.4g of each batch root of Dahurain angelica sample, accurate claim surely, put in the 50ml measuring bottle, add methyl alcohol 45ml, sonicated (power 300W, frequency 50kHz) 1 hour is taken out, and puts coldly, is diluted to scale with methyl alcohol, shakes up, and filters, and gets subsequent filtrate, promptly gets.Accurate each need testing solution 10 μ l that draw inject liquid chromatograph, measure peak area, calculate the content of oxypeucedanin, Imperatorin in each batch sample.See table 41,42.
Table 41 sample source
Table 42 angelica root assay result (n=2)
13 conclusions
Proved that through previous experiments the height of oxypeucedanin content is to judge whether one of sulphuring coumarin kind compound intuitively of the root of Dahurain angelica.This method can be measured oxypeucedanin and Imperatorin simultaneously, and stable, feasible, and this provides a new method for the quality control of the root of Dahurain angelica.From the result of assay, the content of oxypeucedanin nearly all is lower than sensing range in the sulphuring root of Dahurain angelica.Not in the sulphuring root of Dahurain angelica; The average content of oxypeucedanin reaches 0.17%, and the average content of Imperatorin reaches 0.14%, considers the difference in the different places of production; The content of oxypeucedanin must not be lower than 0.12% in the tentative root of Dahurain angelica; Between 0.12-0.23%, the content of Imperatorin must not be lower than 0.1%, between 0.11-0.22%.The effective evaluation angelica root quality that is established as of this method provides scientific basis.
Below further prove beneficial effect of the present invention through pharmacodynamics test.
Test Example 1 angelica root analgesic activity test of the present invention
Adopt the acetic acid twisting method first to the angelica root of sulphuring and sulphuring not totally 6 samples carried out the comparative studies of 3 analgesic activities, for the selection of bulk drug in the quality assessment of angelica root and the compound preparation provides foundation.
Press the method for embodiment 9 and measure, wherein in the sulphuring root of Dahurain angelica oxypeucedanin content be lower than detectability or＜0.02%, Imperatorin 0.08-0.3%: oxypeucedanin 0.12-0.23% in the root of Dahurain angelica of sulphuring not, Imperatorin 0.11-0.22%.
1 experimental section
1.1 experiment material
The healthy Kunming mouse of one-level, Chengdu University of Traditional Chinese Medicine's Experimental Animal Center, the animal conformity certification is No. 7, the real moving pipe in river, 18～22g.Benorylate sheet (original name benoral), Diao Group Chengdu Pharmaceutical Co., Ltd., every 0.5g, lot number: 0311238; Glacial acetic acid pure for analyzing (Chengdu associating chemical reagent research institute); Absolute ethyl alcohol pure (sky, Chengdu China Science and Technology Co., Ltd., lot number: 20040915) for analyzing.Sample source and numbering are seen table 43.
The source of table 43 root of Dahurain angelica and numbering
Above medicinal material is accredited as the dry root of samphire Radix angelicae dahuricae Angelica dahurica (Fisch.ex Hoffm.) Benth.et Hook.f.var.formosana (Boiss.) Shan et Yuan through professor Ma Yuying of Chinese traditional medicine identification teaching and research room of Chengdu University of Traditional Chinese Medicine.
1.2 the preparation method of the root of Dahurain angelica
Get root of Dahurain angelica sample powder 100g, add after an amount of 70% ethanol soaks into 4h, medicinal powder is packed in the percolation vat, use 70% ethanol percolation, collection obtains 600ml solution, and (flow velocity is 0.5mlmin
-1), the concentrating under reduced pressure percolate is to 100ml, and making concentration is 1gml
-1Root of Dahurain angelica alcohol extract.
1.3 the experimental technique of pharmacology and result
1.3.1 mouse acetic acid twisting method test
Get 96 of healthy mices, ♀ ♂ half and half is divided into distilled water group, benoral group (20gkg at random
-1) 2 groups, No. 407 and No. 228 (crude drug 2.5,5,10gkg
-1) 6 groups.Totally 8 groups.Each treated animal is normally raised, in each gastric infusion of 8:30 1 time every day, successive administration 3d.Behind last administration 1h, every ip in mice 0.7% glacial acetic acid solution 0.2ml observes and writes down the number of times (belly shrinks indent, trunk and back leg extension, hips up) of respectively organizing mouse in the injection glacial acetic acid 15min and writhing response occurring immediately.Organize the analgesia inhibiting rate (table 44) that a t checked and be calculated as follows the administration group.
Table 44 sulphuring and sulphuring root of Dahurain angelica sample alcohol extract mouse acetic acid twisting method revision test one result not
(?
n＝12)
Compare with the distilled water group,
*P＜0.05,
*P＜0.01,
* *P＜0.001
1.3.3 mouse acetic acid twisting method revision test two is got 96 of healthy mices, ♀ ♂ half and half is divided into distilled water group, benoral group (20gkg at random
-1) 2 groups, No. 408 (No. 435) (crude drug 2.5,5,10gkg
-1) 6 groups.Totally 8 groups.Method of administration, experimental technique are with under " 1.3.1 " item (table 45).
Table 45 sulphuring and sulphuring root of Dahurain angelica sample alcohol extract mouse acetic acid twisting method revision test two results not
(?
n＝12)
Compare with the distilled water group,
*P＜0.05,
* *P＜0.001
2 discuss
2.1 through adopting mouse acetic acid twisting method to sulphuring and the not comparison of sulphuring angelica root analgesic activity, the result shows, benoral 20gkg
-1Group demonstrates significant analgesia role (P＜0.001); Average writhing response number of times of mouse and distilled water control group are relatively after each sample alcohol extract administration of the root of Dahurain angelica; Three each dose groups of sample of sulphuring do not demonstrate analgesic activity (P＞0.05); And under same test condition, four dose groups of three of sulphuring samples not: No. 405 (crude drug 10gkg
-1Group) (P＜0.01), No. 407 (crude drug 5gkg
-1Group) (P＜0.01), No. 407 (crude drug 2.5gkg
-1Group) (P＜0.05), No. 408 (crude drug 2.5gkg
-1Group) the mouse writhing number of times due to the glacial acetic acid can obviously be reduced in (P＜0.05), and significant difference is all arranged, and demonstrates tangible analgesic effect.
2.2 lot of experiments result shows; Sulphuring all has very big influence to the chemical constitution and the analgesic activity of angelica root; Some countries are to the limit detection of the Chinese crude drug implementation sulphur of China's outlet in addition, and the sulphuring habitat processing method that adopts is now demanded urgently improving, and the standardized study that the root of Dahurain angelica place of production processes is strengthened in suggestion; Adopt suning method or science, convenient and practical job operation more, to guarantee quality of medicinal material and drug safety.
To sum up, adopt mouse acetic acid twisting method to observe mouse and irritate behind stomach sulphuring and the sulphuring Angelice dehurica crude drug alcoholic extract not in the 15min influence for three days on end writhing response.In the analgesic experiment, with the distilled water control group relatively, the sample sets there was no significant difference of sulphuring (P＞0.05), 3 of sulphuring root of Dahurain angelica samples do not cause the writhing response number of times that pain causes to the mouse glacial acetic acid and obviously reduce, and significant difference (P＜0.05) is arranged.The sulphuring angelica root does not demonstrate analgesic effect, and the sulphuring medicinal material does not then demonstrate significant analgesia role.Wherein the content difference with oxypeucedanin is bigger, and this experiment showed, through controlling the quality of oxypeucedanin, can reach the purpose of the quality of control root of Dahurain angelica preparation.
Claims (12)
1. an angelica root, Angelica Dahurica extract or contain the detection method of the pharmaceutical composition of Angelica Dahurica extract, it is characterized in that: its is that index components detects with oxypeucedanin, Imperatorin simultaneously, and it comprises the steps:
A, through high effective liquid chromatography for measuring, the ultraviolet detection wavelength is 305nm;
B, get oxypeucedanin, Imperatorin reference substance, add methyl alcohol and process reference substance solution;
The preparation of c, need testing solution: get angelica root, extract or preparation, add methyl alcohol, power 300W, the ultrasonic Extraction 0.5-1.5 of frequency 50kHz hour, take out, put coldly, be diluted to scale with methyl alcohol, shake up, filter, get subsequent filtrate, promptly get;
D, accurate reference substance solution and the need testing solution injection liquid chromatograph drawn of difference are measured, and promptly get;
Wherein, the chromatographic condition of the said high performance liquid chromatography of a step is: chromatographic column: Diamonsil
TMC
18The HPLC chromatographic column, 250 * 4.6mm, 5 μ m; Moving phase: methyl alcohol-tetrahydrofuran-water=56: 9: 35; Flow velocity: 1.0ml/min; Column temperature: 30 ℃; Sample size: 10 μ l; Detect wavelength: 305nm; Number of theoretical plate should be not less than 4000 in oxypeucedanin, Imperatorin.
2. detection method according to claim 1 is characterized in that: the content of oxypeucedanin must not be lower than 0.12%w/w in the described angelica root, and the content of Imperatorin must not be lower than 0.1%w/w.
3. detection method according to claim 2 is characterized in that: contain oxypeucedanin 0.12～0.23%w/w in the described angelica root, Imperatorin: 0.11～0.22%w/w.
4. detection method according to claim 1 is characterized in that: contain oxypeucedanin 9.50～14.24mg/g in the said Angelica Dahurica extract.
5. detection method according to claim 4 is characterized in that: also contain Imperatorin 5.52～8.28mg/g in the said Angelica Dahurica extract.
6. according to claim 4 or 5 described detection methods, it is characterized in that: said Angelica Dahurica extract is the water or the extractive with organic solvent of angelica root.
7. detection method according to claim 6 is characterized in that: said Angelica Dahurica extract is to be prepared from following method: get the angelica root meal, add 50～95% alcohol extracts; Extract promptly gets Angelica Dahurica extract through 60～70 ℃ of concentrating under reduced pressure dryings.
8. detection method according to claim 7 is characterized in that: described alcohol extract method is percolation or ultrasonic extraction.
9. detection method according to claim 4 is characterized in that: the HPLC finger-print of described Angelica Dahurica extract is as shown in Figure 2, and chromatographic condition is: chromatographic column: Diamonsil
TMC
18The HPLC chromatographic column, 250 * 4.6mm, 5 μ m; Moving phase: methyl alcohol-tetrahydrofuran-water=56: 9: 35; Flow velocity: 1.0ml/min; Column temperature: 30 ℃; Sample size: 10 μ l; Detect wavelength: 305nm; Number of theoretical plate should be not less than 4000 in oxypeucedanin, Imperatorin.
10. detection method according to claim 4 is characterized in that: described pharmaceutical composition is to be active component with the Angelica Dahurica extract, adds the medicament that acceptable accessories or complementary composition are prepared from.
11. detection method according to claim 10 is characterized in that: described medicament is capsule, tablet, granule, pill or oral liquid.
12. detection method according to claim 10 is characterized in that: described medicament is soft capsule or pill.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010622736 CN102058641B (en) | 2009-12-29 | 2010-12-28 | Detection method of angelica dahurica, extract and pharmaceutical composition containing angelica dahurica extract |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910265021.5 | 2009-12-29 | ||
| CN200910265021 | 2009-12-29 | ||
| CN 201010622736 CN102058641B (en) | 2009-12-29 | 2010-12-28 | Detection method of angelica dahurica, extract and pharmaceutical composition containing angelica dahurica extract |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102058641A CN102058641A (en) | 2011-05-18 |
| CN102058641B true CN102058641B (en) | 2012-12-26 |
Family
ID=43994266
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010622736 Expired - Fee Related CN102058641B (en) | 2009-12-29 | 2010-12-28 | Detection method of angelica dahurica, extract and pharmaceutical composition containing angelica dahurica extract |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102058641B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104523783B (en) * | 2014-12-11 | 2017-11-03 | 黑龙江仁合堂药业有限责任公司 | The big production extraction process of Angelica Dahurica extract in angelica root |
| CN108152392B (en) * | 2017-12-08 | 2020-09-08 | 宋瑞 | Detection method of angelica dahurica coumarin in veterinary medicine |
| CN109568359A (en) * | 2018-02-06 | 2019-04-05 | 广东方制药有限公司 | A kind of preparation method of angelica dahurica root dispensing granule |
| CN108619183A (en) * | 2018-06-04 | 2018-10-09 | 西安交通大学 | The simple extraction methods of coumarin kind compound and its application in a kind of root of Dahurain angelica |
| CN108815156B (en) * | 2018-07-10 | 2020-04-28 | 南通大学 | Application of oxypeucedanin in preparing medicine for treating postoperative pain |
| CN109045025B (en) * | 2018-07-23 | 2020-05-08 | 南通大学 | New use of oxidized decursin |
| CN109406681B (en) * | 2018-12-21 | 2021-11-16 | 广东一方制药有限公司 | Construction method and detection method of UPLC characteristic spectrum of radix angelicae medicinal material |
| CN114295759B (en) * | 2021-12-31 | 2023-10-24 | 成都中医药大学 | Quality detection method of herba asari or herba asari extract for treating migraine |
-
2010
- 2010-12-28 CN CN 201010622736 patent/CN102058641B/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| 高颖.白芷配方颗粒质量标准的规范化研究.《中国优秀硕士学位论文全文数据库 医药卫生科技辑》.2009,(第7期),E057-277. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102058641A (en) | 2011-05-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102058641B (en) | Detection method of angelica dahurica, extract and pharmaceutical composition containing angelica dahurica extract | |
| CN102138998B (en) | Honey tree fruit general flavone extract and application thereof in preparation of medicaments and food for resisting fatigue, hypoxia or plateau hypoxia | |
| CN104922196B (en) | The preparation of small pagodatree flower general flavone extract and quality determining method | |
| CN101596275A (en) | The method of quality control of Radix Ginseng in the YIXINSHU Chinese medicine preparation | |
| CN102977114B (en) | Inoscavin A as a monomeric component in phellinus as well as prepearation method and application thereof | |
| CN102335260A (en) | Processing principle-based individualized and characteristic quality evaluation method for Gardenia jasminoides Ellis decoction pieces | |
| CN103340938A (en) | Preparation method for tartary buckwheat standard extract FT83 and high pure quercetin | |
| CN101337060B (en) | Quality control method of Gongyankang grannule | |
| CN102488819A (en) | Preparing method for daylily flower extract | |
| CN102539599B (en) | Method for detecting liver-enhancing medicine | |
| CN101810696B (en) | Total triterpenoids of apples and method for preparing same | |
| CN102068657B (en) | Quality control method of venation relaxing pill as Chinese herbal preparation | |
| CN102000066B (en) | Inula helianthus-aquatica extract, anti-tumor medicament using same as active ingredient, preparation method and application thereof | |
| CN103575821A (en) | Detection method of 14 chemical components in Tangminling preparation | |
| CN105106252A (en) | Method for extracting and separating flavonoid compounds from hypericum japonicum | |
| CN102397260B (en) | Method for preparing sinomenine hydrochloride tablet | |
| CN101766664B (en) | Detection method of total saponin of Radix Ilicis Asprellae | |
| CN103113434B (en) | Method for preparing monoterpene glycoside from oil peony seed cake | |
| CN107607665A (en) | One seedling medicine subprostrate sophora and its Chinese medicine preparation method of quality control | |
| CN103575823A (en) | Detection method of 8 chemical components in Tangminling preparation | |
| CN102397264B (en) | Method for preparing sinomenine hydrochloride sustained-release tablet | |
| CN102764320A (en) | Psychotria sp. extract, and preparation method and antineoplastic application thereof | |
| CN101176755B (en) | Application of meletin-7-0-glycoside in mass control of cudrania tricuspidata or preparations thereof | |
| CN100565202C (en) | The detection method of medicine containing swertia psedochinensis and milk thistle silybum marianum | |
| CN103316087B (en) | Semen Myristicae five tastes effective site and preparation method thereof, quality determining method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20121226 Termination date: 20151228 |
|
| EXPY | Termination of patent right or utility model |