CN102058641A - Angelica dahurica extract and quality detection method - Google Patents
Angelica dahurica extract and quality detection method Download PDFInfo
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- CN102058641A CN102058641A CN 201010622736 CN201010622736A CN102058641A CN 102058641 A CN102058641 A CN 102058641A CN 201010622736 CN201010622736 CN 201010622736 CN 201010622736 A CN201010622736 A CN 201010622736A CN 102058641 A CN102058641 A CN 102058641A
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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 Radix Angelicae Dahuricae extract and quality determining method, belong to drug world.
Background technology
Radix Angelicae Dahuricae beginning is stated from Shennong's Herbal, for commonly used induce sweat, analgesic.The Radix Angelicae Dahuricae is the main flow kind of Radix Angelicae Dahuricae commodity medical material, accounts for 70% of national commodity Radix Angelicae Dahuricae output.At present report utilizes the document of the Radix Angelicae Dahuricae more, as 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 Radix Angelicae Dahuricae, extracts.The selectivity basis that this extract comprises is imperatorin, isoimperatorin, 5-hydroxyl-8-methoxyl group psoralen, bergapton, or its any or several combination.This extract also comprises any in the compositions such as Radix Angelicae Dahuricae element, aqua oxidation peucedanin, different oxypeucedanin, 5-methoxyl group-8-hydroxyl psoralen, xanthotoxol or several combination.This extract can be by solvent extraction method, solvent extraction, macroporous adsorbent resin method, lead salt precipitation, column chromatography, liquid-any one methods such as liquid adverse current partography, 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, isoimperatorin, three kinds of compositions of bergapton accounts for 5～100% (w/w) of whole total coumarins content.
The habitat processing method of the Radix Angelicae Dahuricae is bigger to Radix Angelicae Dahuricae quality influence, the main at present sulfur fumigation that adopts, still, its coumarin and volatile oil composition reduce greatly behind a large amount of experiment confirm sulphurings, the drug effect instability of the Radix Angelicae Dahuricae, it is most important therefore to control the angelica root quality.Report about Radix Angelicae Dahuricae method of quality control has: " Chinese pharmacopoeia version in 2000 is only limited to former plant variety, medical material character 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, Deng, the comparison of imperatorin content assaying method in the Radix Angelicae Dahuricae, 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 methanol: water (70: 30) is mobile 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 Radix Angelicae Dahuricae HPLC finger printing, World Science technology-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, cluster analysis and similarity evaluation to estimate, and different tests project sample in the Radix Angelicae Dahuricae standardized planting has been carried out the comparison of finger printing.The result: sulphuring and not the finger printing of sulphuring sample exist than big-difference, set up the sulphuring and the finger printing of sulphuring angelica root not respectively.At present report quantitatively above-mentioned or qualitatively method can't effectively control the quality of the Radix Angelicae Dahuricae.
Summary of the invention
Technical scheme of the present invention has provided a kind of Radix Angelicae Dahuricae extract, and another technical scheme of the present invention has provided the quality determining method of angelica root and Radix Angelicae Dahuricae extract.
The invention provides a kind of Radix Angelicae Dahuricae extract, it contains oxypeucedanin 9.50-14.24mg/g.It also contains imperatorin 5.52-8.28mg/g.
Radix Angelicae Dahuricae extract of the present invention is the water or the extractive with organic solvent of angelica root.It is to be prepared from by following method: get the angelica root coarse powder, add the 50-95% ethanol extraction; Extracting solution promptly gets Radix Angelicae Dahuricae extract through 60-70 ℃ of concentrating under reduced pressure drying.Wherein, described ethanol extraction method is percolation or ultrasonic extraction.
The HPLC finger printing of described Radix Angelicae Dahuricae extract as shown in Figure 2, chromatographic condition is: chromatographic column: Diamonsil
TMC
18HPLC chromatographic column (250 * 4.6mm, 5 μ m); Mobile phase: methanol-oxolane-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 Radix Angelicae Dahuricae of the present invention or Radix Angelicae Dahuricae extract, be meant the Radix Angelicae Dahuricae without sulphuring, wherein, the Radix Angelicae Dahuricae is the root of Umbelliferae archangel Radix Angelicae Dahuricae 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 Radix Angelicae Dahuricae 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 drop pill.
The invention provides a kind of method for quality that detects angelica root, Radix Angelicae Dahuricae extract or contain the pharmaceutical composition of Radix Angelicae Dahuricae 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, by high effective liquid chromatography for measuring, the ultraviolet detection wavelength is 305nm;
B, get oxypeucedanin, imperatorin reference substance, add methanol and make reference substance solution;
The preparation of c, need testing solution: get angelica root, extract or preparation, add methanol, power 300W, the supersound extraction 0.5-1.5 of frequency 50kHz hour, take out, put coldly, be diluted to scale with methanol, shake up, filter, get subsequent filtrate, promptly;
D, accurate reference substance solution and the need testing solution injection chromatograph of liquid drawn of difference are measured, promptly.
Wherein, the described chromatographic condition of a step is: chromatographic column: Diamonsil
TMC
18HPLC chromatographic column (250 * 4.6mm, 5 μ m); Mobile phase: methanol-oxolane-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 by detecting 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 medical material, be used for detecting, differentiate the sulphuring and the sulphuring Radix Angelicae Dahuricae not, can monitor the quality of Radix Angelicae Dahuricae medicine effectively, the drug effect of medicine of the present invention is clear and definite, and controllability is stronger.
Description of drawings
Fig. 1 Radix Angelicae Dahuricae extraction process of the present invention flow chart
(mobile phase is methanol 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 (crude drug is sulphuring not, lot number 091001)
Fig. 5 test sample chromatogram (crude 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 (mobile phase is methanol-water 70: 30)
Figure 12 need testing solution chromatogram (the not sulphuring Radix Angelicae Dahuricae)
Figure 13 need testing solution chromatogram (the sulphuring Radix Angelicae Dahuricae)
Figure 14 imperatorin reference substance chromatogram
Figure 15 need testing solution 60min chromatogram
The specific embodiment
The preparation method of embodiment 1 Radix Angelicae Dahuricae extract
Angelica root breaks into coarse powder, with the speed percolation of 2.5ml/minkg, collects the medicinal liquid of 6 times of amounts behind the 50% alcohol solution dipping 24h, carries out the concentrating under reduced pressure molding under 60～70 ℃ temperature, and drying gets Radix Angelicae Dahuricae extract of the present invention.
The preparation of embodiment 2 Radix Angelicae Dahuricae extracts of the present invention
The angelica root coarse powder adds 70% ethanol of 12 times of amounts, power 80W, and supersound extraction 50min under 55 ℃ of temperature extracts 3 times, and method for concentration is drying to obtain Radix Angelicae Dahuricae extract of the present invention with embodiment 1.
The preparation of embodiment 3 Radix Angelicae Dahuricae extracts of the present invention
Get angelica root, extract, concentrate by embodiment 1 method, the medicinal liquid of gained carries out spray drying.
Spray-dired condition is: the feed liquor temperature of charge: 70～80 ℃; Extractum relative density: 1.08～1.10 (60 ℃); Feed liquor speed: 60～70ml/min; Inlet temperature: 138～140 ℃; Leaving air temp: 83～85 ℃.Get Radix Angelicae Dahuricae 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 Radix Angelicae Dahuricae extract that embodiment 1 prepares, add acceptable accessories, prepare tablet.
The preparation of embodiment 6 medicines of the present invention
Get the medicinal liquid that embodiment 1 concentrating under reduced pressure obtains, after the remove impurity, add acceptable accessories, prepare oral liquid.
Embodiment 1-6 is to the preparation method of Radix Angelicae Dahuricae extract of the present invention and medicine; should not be construed as is limiting the scope of the 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.
One, set up index investigation method
The Coumarins composition is its main active in the Radix Angelicae Dahuricae, multiple pharmacological effect such as modern pharmacology experiment shows that furocoumarin contained in the Radix Angelicae Dahuricae has antipyretic-antalgic, antiinflammatory, relievings asthma, blood pressure lowering, antibiotic, spasmolytic, photosensitive, activation sympathetic system hormone.In addition, imperatorin is one of its main effective ingredient.So determine with total coumarins, imperatorin to be chemical index.For taking into account molding, with the reference index of dried cream rate as technical study.
1. the mensuration of dried cream rate
Precision pipettes each extracting solution 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. total coumarins Determination on content
With the imperatorin is reference substance, adopts spectrophotography to measure at the 300nm place.Standard 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 response rate is 98.25%, RSD=1.70%.
3. imperatorin Determination on content
Adopt high effective liquid chromatography for measuring.Chromatographic condition: with octadecyl silane is filler; Methanol: water (70: 30) is mobile phase; The detection wavelength is 300nm.Column temperature: 30 ℃, flow velocity is 1.0ml/min, sample size: 10 μ L.
Standard 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: the sample test solution is stable in 120h, RSD=2.35%; The method response rate is 98.68%, RSD=1.96%.
Two, Radix Angelicae Dahuricae extract Study on Preparation of the present invention
1. extraction process is investigated
In the preliminary experiment Radix Angelicae Dahuricae 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 as the elasticity of fitted tube, the addition of fresh liquor, the uncertain factors such as thickness of Cotton Gossypii in the fitted tube process, finally selects single factor to investigate.
(1) extraction solvent is investigated
Take by weighing two parts of 100g Radix Angelicae Dahuricae coarse powder, water and 95% ethanol extract respectively, and extracting solution concentrates and is settled to certain volume, press the content of total coumarins and imperatorin in content assaying method calculating water extract and the alcohol extract.The investigation result of table 1 extraction solvent
(2) investigation of medical material granularity
Take by weighing each 3 parts of the coarse powder of angelica root, coarse powder, middle powder, every part of 100g, the ethanol swelling with 50% is after 1 hour, fitted tube flooded after 4 hours, with the speed percolation extraction of 0.25ml/min, collect percolate 600ml sampling, measure coumarin, imperatorin, dried cream rate respectively, the results are shown in Table 2:
The investigation result of table 2 medical material granularity
The result shows: medical material is best with the effect of coarse powder percolation, and medicated powder is thick excessively, and effective ingredient is not easy to leach, and pulverizing medicinal materials gets too thin, and medicinal liquid is difficult for flowing out, blockage phenomenon often in the amplification test.Determined that thus with coarse powder be best grinding particle size.
(3) Different Extraction Method is extracted the result relatively
Get 3 parts of Radix Angelicae Dahuricae coarse powder, 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 percolation 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 supersound extraction 30min, and 2 times, each collects 6 times of medicine amount liquids.
The investigation result of table 3 extracting method
As seen from table: percolation is better than other extracting method to the extraction effect of index components, and so selects percolation to extract.
The comprehensive above result that investigates determines the process route with ethanol percolate extraction.
(4) investigation of solvent concentration
Take by weighing 5 parts of the coarse powder 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 percolation 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 total coumarins, 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 by to 50% and 70% alcoholic acid comparison.
(5) investigation of percolation speed
Take by weighing 4 parts of the coarse powder of angelica root, every part of 100g, 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 percolation 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 percolation speed
The result shows: the percolation VELOCITY EXTRACTION effect of 0.25ml/min is better than other infiltration rate.So determine that the percolation speed of 100g medical material is 0.25ml/min, promptly percolation speed is 2.5ml/minkg.
(6) investigation of soak time
Take by weighing 4 parts of the coarse powder 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 percolation of 0.25ml/min, collect percolate 600ml.
The investigation result of table 6 soak time
The result shows: soak 24h extraction effect the best, determine that soak time is 24h.
(7) investigation of collecting amount
Take by weighing the coarse powder 100g of angelica root, the ethanol swelling with 50% is after 1 hour, fitted tube, soak 24h after, extract with the speed percolation 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 substantially, and determine that collecting amount is 600ml.Consider to amplify and produce,,, when guaranteeing that most effective ingredient ooze out, also shortened the production cycle like this, saved 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, determined optimum extraction process: angelica root breaks into coarse powder, with the speed percolation of 2.5ml/minkg, collects the medicinal liquid 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 total coumarins, imperatorin content and the dried cream rate of above-mentioned sample liquid respectively, the results are shown in 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 total coumarins is index (in conjunction with a 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 total coumarins.
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 molding easily adds the consideration of producing for to amplification, so decision wouldn't be selected the chemical subtraction method for use, we have carried out the investigation of centrifugal remove impurity to percolate for this reason.
Get two parts of the percolates that extract by set technology, a directly centrifugal with medicinal liquid; Another part medicinal liquid is evaporated to every ml medicinal liquid and is equivalent to centrifugal (5000r/min, centrifugal 1h) behind the 0.5g crude drug.The result is as follows:
Table 9 centrifugal method is investigated the result
The centrifugal method result
Medicinal liquid is centrifugal poor effect directly, and medicinal liquid is not seen layering
Medicinal liquid concentrates the centrifugal medicinal liquid 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 after centrifugal and centrifugal gained precipitation are carried out imperatorin and dried cream rate, the result is as follows:
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 concentrated solution, and imperatorin is a liposoluble constituent, and so dissolving hardly in aqueous solution is the precipitation of formation.
So determine percolate is directly concentrated molding.
3. concentration technology research
Angelica dahurica coumarin constituents case of thermal instability, relatively comprehensive, the method for selection concentrating under reduced pressure.Be necessary that at first the heat stability of extracting solution is investigated when concentrating, method is as follows: experimental technique and result: get 3 parts of extracting solution, 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 as follows:
Table 11 Radix Angelicae Dahuricae heat stability is investigated the result
Processing method 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 Radix Angelicae Dahuricae 80 ℃ following 4 hours with interior more stable, heated time is long, causes effective ingredient to decompose easily, content reduces.Because concentrating under reduced pressure efficient height keeps 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.Should under 60～70 ℃ temperature, carry out concentrating under reduced pressure so determine this product.
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, few to thermal sensitivity composition destruction, production process is simple, and is easy and simple to handle, be easy to control of quality, be applicable to industrial continuous big production, it is few that institute adds adjuvant, 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 extractum relative density, adjuvant and consumption, feed liquor temperature of charge, charging rate, inlet temperature, leaving air temp etc.
(1) investigation of different auxiliary material
Consider cost and adaptability, dextrin, soluble starch, three kinds of adjuvants of lactose are investigated.Extracting solution after medical material concentrated is divided into 4 equal portions; Press 50% of dry extract respectively and add 3 kinds of adjuvants, make dissolving or be uniformly dispersed; 1 part does not add any adjuvant in addition, spray-dried separately behind the abundant mixing, and the result is as follows:
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 extractum after adding, and has precipitation to produce; Lactose high temperature is easily softening, and material is flow-like and adheres to inwall when dry.So selecting dextrin is its spray-dired adjuvant.
(2) investigation of adjuvant dextrin consumption:
According to this product Study on extraction process, determined with 50% alcoholic solution as extraction solvent.The extractum spray drying of alcohol extraction is difficulty, and because of its stickiness is big, in spray-dired process, have the adjuvant that resists glutinous and peptizaiton normal the adding, to improve the character of material.In conjunction with this product situation, the dextrin addition is investigated during to this product drying in the experiment, and the result is as follows.
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
By the result as can be known, when the dextrin consumption is 40% when above of dried cream amount in the extractum, 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 determining spray drying.
(3) investigation of extractum relative density
The extractum (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 (inlet temperature: 138～140 ℃, leaving air temp: 82～85 ℃), observes the dry materials situation, and the result is as follows:
The screening of table 14 extractum relative density
Extractum 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 extractum relative density is chosen in 1.08～1.10 (60 ℃) can get quality powdered extract powder preferably when carrying out spray drying.The extractum relative density is too high, and the extractum viscosity is big, easy blocking pipe, and the atomizing difficulty, and the granule that atomizing forms is big, and material easily coheres, discharging color burn, even the burnt phenomenon of sticking with paste of appearance; 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 determine that the extractum relative density is 1.08～1.10 (60 ℃).
(4) investigation of inlet temperature, leaving air temp
To contain extractum (1.10,60 ℃ of the relative densities) charging of the dextrin of dried cream amount 50%, under different inlet temperature, leaving air temp, carry out spray drying respectively, observe the dry materials situation.The result is as follows.
The investigation of table 15 inlet temperature, leaving air temp
By above-mentioned experimental result as can be known, inlet temperature is 133～135 ℃, glues wall when leaving air temp is 80～82 ℃ on a small quantity, a small amount of block is arranged; Inlet temperature is 138～140 ℃, when leaving air temp is 83～85 ℃, effect is better, obtains yellow loose powdered; Inlet temperature 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 determine inlet temperature, leaving air temp.
(5) determining of charging fluid temperature
Material need keep uniform temperature in the past entering the spray tower drying, helped reducing the viscosity of material, made the moisture back evaporation rapidly in entering drying tower in the material, thereby shortened drying time, improved drying efficiency.
Investigate result's (investigate early stage) as can be known by Radix Angelicae Dahuricae heat stability in this product concentration technology, this product 80 ℃ 4 hours with interior more stable.For taking into account drying efficiency and index components, determine 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 nebulizer, 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 as follows:
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, determines that charging rate is 60～70ml/min.Also should do corresponding adjustment in the production according to the inherent 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 as follows:
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 ingredient 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 content
By " 2005 editions one appendix IX H of Chinese pharmacopoeia aquametry, first method is measured the water content of three batches of spray-dried powders, and the result is as follows.
The measurement result of table 18 spray-dried powders water content
The result shows: the dry extract water content that spray drying obtains is less.
Comprehensive above-mentioned result of study, determine that the spray drying condition is:
The addition of adjuvant dextrin: 40% of dried cream amount; Feed liquor temperature of charge: 70～80 ℃; Extractum relative density: 1.08～1.10 (60 ℃); Feed liquor speed: 60～70ml/min; Inlet temperature: 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 Radix Angelicae Dahuricae extract preparation technology, process chart is as shown in Figure 1.
The content of oxypeucedanin, imperatorin in the medical material Radix Angelicae Dahuricae 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 mobile phase, the separating degree of oxypeucedanin is still not good in the sample, sees Fig. 2 with methanol-water (65: 35).After adjusting mobile phase (methanol-oxolane-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)
Mobile phase: methanol-oxolane-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, list of references is intended investigating with methanol, ethyl acetate, 95% ethanol, is carried out the investigation of extraction solvent simultaneously.
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 methanol, 95% ethanol, each 45ml of ethyl acetate respectively, supersound process (power 300W, frequency 50kHz) 1 hour, filter, residue washs with corresponding solvent, filters.Filtrate water-bath Back stroke adds methanol and is diluted to 50ml, promptly after doing.With 0.45 μ m filtering with microporous membrane, draw 10 μ l before the sample introduction, inject chromatograph of liquid, 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, the results are shown in Table 19.
The investigation of table 19 extraction solvent
Above result shows that the methanol extraction effect is better, therefore selects methanol as extraction solvent.
3.2 the investigation of extracting method
Relatively supersound extraction, reflux, extract,, merceration 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 methanol 45ml, supersound process (power 300W, frequency 50kHz) 1 hour.Portion is put in the 100ml conical flask, adds methanol 45ml, leaves standstill 24 hours.Portion is put in the 100ml round-bottomed flask, adds methanol 45ml, reflux, extract, 1 hour.Each extracting solution filters, and is diluted in the 50ml measuring bottle with methanol, shakes up, and filters, and gets subsequent filtrate, promptly.With 0.45 μ m filtering with microporous membrane, draw 10 μ l before the sample introduction, inject chromatograph of liquid, measure the content of imperatorin in the calculation sample according to aforementioned chromatographic condition.The method operation repetitive is twice like this.The results are shown in Table 20.
Table 20 Different Extraction Method extraction effect relatively
The result shows that the reflux, extract, method is relatively poor, and supersound extraction and merceration extract better, consider that the merceration time is oversize, therefore selects ultrasonic extracting method for use.
3.3 the investigation of supersound extraction time
Get 3 parts of medicines of the present invention (091002 batch), every part of 0.2g, the accurate title, decide, put in the 50ml measuring bottle, add methanol 45ml, 0.5 hour, 1 hour, 1.5 hours each extracting solution of supersound process (power 300W, frequency 50kHz) take out, put cold,, shake up to scale with methanol constant volume, filter, get subsequent filtrate, promptly.With 0.45 μ m filtering with microporous membrane, draw 10 μ l before the sample introduction, inject chromatograph of liquid, measure the content of imperatorin in the calculation sample according to aforementioned chromatographic condition.The method operation repetitive is twice like this.The results are shown in Table 21.
Extraction effect of different supersound 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.
In summary, 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 methanol 45ml, supersound process (power 300W, frequency 50kHz) 1 hour is taken out, and puts coldly, is diluted to scale with methanol, shake up, filter, get subsequent filtrate, promptly.
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 chromatography, measure peak area by above-mentioned chromatographic condition.The results are shown in Table 22.With the peak area is vertical coordinate, and the amount of oxypeucedanin is an abscissa, 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 chromatography, measure peak area by above-mentioned chromatographic condition.The results are shown in Table 23.With the peak area is vertical coordinate, and the amount of imperatorin is an abscissa, 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, the results are shown in Table 23.
Table 23 precision is investigated the result
Measurement 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, and measurement result sees Table 24.
Measurement 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 results are shown in Table 25.
The stability test of table 25 need testing solution
Measurement result shows: need testing solution place in 24 hours basicly stable.
7, repeatability 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 results are shown in Table 26.
The test of table 26 repeatability
Measurement result shows: oxypeucedanin, imperatorin element contain heavily in this method working sample, and repeatability 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 results are shown in Table 27,28.
Table 27 oxypeucedanin response rate experimental result
Table 28 imperatorin response rate experimental result
Measurement result shows: this method has the response rate 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 methanol 45ml, supersound process (power 300W, frequency 50kHz) 1 hour is taken out, and puts coldly, is diluted to scale with methanol, shakes up, and filters, and gets subsequent filtrate, promptly.Accurate each need testing solution 20 μ l that draw inject chromatograph of liquid, measure peak area, calculate the content of imperatorin in each batch sample.See Table 29.
Table 29 assay result (n=2)
By above measurement result as seen: 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 adjuvant amount is 40% of a dried cream amount in known the invention described above medicine, just adjuvant accounts for 29% of total extract, and dried cream accounts for 71% of total extract, therefore, oxypeucedanin in the Radix Angelicae Dahuricae extract, the content of imperatorin can be converted by last table result and obtain, and the results are shown in Table 30.
After the conversion, before and after the oxidation of 10 batches of Radix Angelicae Dahuricae 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 Radix Angelicae Dahuricae 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 crude 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.As seen, be that oxypeucedanin content can't be measured in the prepared extract of raw material with the sulphuring Radix Angelicae Dahuricae, 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
Diode array detector Waters 2996 types
Electronic analytical balance BP121S (Beijing Sartorius balance company limited)
Oxypeucedanin reference substance (the HPLC area normalization method is determined 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 methanol, oxolane are chromatographically pure (German Fisher), and water is double distilled water, and all the other reagent are analytical pure.
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 petroleum ether-ether (5: 3), chloroform-methanol (100: 2), three kinds of development systems of petroleum ether-acetone (5: 1) to launch respectively, sulphuric acid-ethanol colour developing all is single speckle in the thin layer chromatography; Experience under ultra-violet lamp (365nm) all is the yellow fluorescence speckle.
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, inspect under 310nm, not seeing has impurity peaks to occur.See Fig. 8.
3 detect the selection of wavelength
Get the oxypeucedanin reference substance and make the solution that contains oxypeucedanin 10.7 μ g/ml, get the imperatorin reference substance and make 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 wave-length coverage.Imperatorin has an absworption peak at 301nm wavelength around place, and oxypeucedanin has an absworption peak at 310nm wavelength around place, and peak shape changes mild, is convenient to quantitative measurement, finally selects 305nm as detecting wavelength.The results are shown in Figure 9,10.
4, the selection of chromatographic condition
Adopt that " the Chinese pharmacopoeia version Radix Angelicae Dahuricae in 2005 the item chromatographic condition of regulation is down investigated, and finds that with methanol-water (55: 45) be mobile phase when carrying out eluting, 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 mobile 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 separating degree of oxypeucedanin does not reach requirement with this understanding.After mobile phase was adjusted into methanol-oxolane-water (56: 9: 35), the oxypeucedanin separating degree 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 may 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)
Mobile phase: methanol-oxolane-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, list of references is intended investigating with methanol, ethyl acetate, 95% ethanol, is carried out the investigation of extraction solvent simultaneously.
Get 3 parts of angelica roots, every part of 0.4g, the accurate title, decide, and puts in the 50ml measuring bottle, adds methanol, 95% ethanol, each 45ml of ethyl acetate respectively, and supersound process (power 300W, frequency 50kHz) 1 hour filters, and residue washs with corresponding solvent, filters.Filtrate water-bath Back stroke adds methanol and is diluted to 50ml, promptly after doing.With 0.45 μ m filtering with microporous membrane, draw 10 μ l before the sample introduction, inject chromatograph of liquid, 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, the results are shown in Table 31.
The investigation of table 31 extraction solvent (n=2)
Above result shows that the methanol extraction effect is better, therefore selects methanol as extraction solvent.
6.2 the investigation of extracting method
Relatively supersound extraction, reflux, extract,, merceration 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 methanol 45ml, supersound process (power 300W, frequency 50kHz) 1 hour.Portion is put in the 50ml conical flask, adds methanol 45ml, leaves standstill 24 hours.Portion is put in the 50ml round-bottomed flask, adds methanol 45ml, reflux, extract, 1 hour.Each extracting solution filters, and is diluted in the 50ml measuring bottle with methanol, shakes up, and filters, and gets subsequent filtrate, promptly.With 0.45 μ m filtering with microporous membrane, draw 10 μ l before the sample introduction, inject chromatograph of liquid, measure the content of imperatorin in the calculation sample according to aforementioned chromatographic condition.The method operation repetitive is twice like this.The results are shown in 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 supersound 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 methanol 45ml, 0.5 hour, 1 hour, 1.5 hours each extracting solution of supersound process (power 300W, frequency 50kHz) take out, put cold, with methanol constant volume to scale, shake up, filter, get subsequent filtrate, promptly.With 0.45 μ m filtering with microporous membrane, draw 10 μ l before the sample introduction, inject chromatograph of liquid, measure the content of imperatorin in the calculation sample according to aforementioned chromatographic condition.The method operation repetitive is twice like this.The results are shown in Table 33.
Extraction effect of different supersound 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.
In summary, 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 methanol 45ml, supersound process (power 300W, frequency 50kHz) 1 hour is taken out, and puts coldly, is diluted to scale with methanol, shake up, filter, get subsequent filtrate, promptly.
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 chromatography, measure peak area by above-mentioned chromatographic condition.The results are shown in Table 34.With the peak area is vertical coordinate, and the amount of oxypeucedanin is an abscissa, 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 chromatography, measure peak area by above-mentioned chromatographic condition.The results are shown in Table 34.With the peak area is vertical coordinate, and the amount of imperatorin is an abscissa, 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, the results are shown in Table 35.
Measurement 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, and measurement result sees Table 36.
The stability test of table 36 reference substance solution
Measurement 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 results are shown in Table 37.
The stability test of table 37 need testing solution
Measurement result shows: need testing solution place in 24 hours basicly stable.
The test of 10 repeatability
Get parallel 5 parts of the Radix Angelicae Dahuricae, 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 results are shown in Table 38.
The test of table 38 repeatability
Measurement result shows: oxypeucedanin, imperatorin cellulose content in this method working sample, repeatability 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 results are shown in Table 39,40.
Measurement result shows: this method has the response rate preferably.
Table 39 oxypeucedanin response rate experimental result
Table 40 imperatorin response rate experimental result
The mensuration of 12 samples
Get each about 0.4g of each batch Radix Angelicae Dahuricae sample, accurate claim surely, put in the 50ml measuring bottle, add methanol 45ml, supersound process (power 300W, frequency 50kHz) 1 hour is taken out, and puts coldly, is diluted to scale with methanol, shakes up, and filters, and gets subsequent filtrate, promptly.Accurate each need testing solution 10 μ l that draw inject chromatograph of liquid, 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 by previous experiments the height of oxypeucedanin content is to judge whether one of sulphuring coumarin kind compound intuitively of the Radix Angelicae Dahuricae.This method can be measured oxypeucedanin and imperatorin simultaneously, and stable, feasible, and this quality control for the Radix Angelicae Dahuricae provides a new method.From the result of assay, the content of oxypeucedanin nearly all is lower than detection range in the sulphuring Radix Angelicae Dahuricae.Not in the sulphuring Radix Angelicae Dahuricae, the average content of oxypeucedanin reaches 0.17%, the average content of imperatorin reaches 0.14%, consider the difference in the different places of production, the content of oxypeucedanin must not be lower than 0.12% in the tentative Radix Angelicae Dahuricae, 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 by pharmacodynamics test.
The 1 angelica root analgesic activity test of the present invention of test example
Adopt the acetic acid twisting method first to the angelica root of sulphuring and sulphuring not totally 6 samples carried out the comparative study of 3 analgesic activities, for the selection of crude drug in the quality evaluation of angelica root and the compound preparation provides foundation.
Press the method for embodiment 9 and measure, wherein in the sulphuring Radix Angelicae Dahuricae oxypeucedanin content be lower than detectability or＜0.02%, imperatorin 0.08-0.3%: oxypeucedanin 0.12-0.23% in the Radix Angelicae Dahuricae 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 quality certification is No. 7, the real moving pipe in river, 18～22g.Benorilate tablet (original name benorylate), Diao Group Chengdu Pharmaceutical Co., Ltd., every 0.5g, lot number: 0311238; Glacial acetic acid is analytical pure (a Chengdu associating chemical reagent institute); Dehydrated alcohol is analytical pure (sky, Chengdu China Science and Technology Co., Ltd., a lot number: 20040915).Sample source and numbering see Table 43.
The source of table 43 Radix Angelicae Dahuricae and numbering
Above medical 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 TCD identification teaching and research room of Chengdu University of Traditional Chinese Medicine.
1.2 the preparation method of the Radix Angelicae Dahuricae
Get Radix Angelicae Dahuricae 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
-1Radix Angelicae Dahuricae alcohol extract.
1.3 the experimental technique of pharmacology and result
1.3.1 mice acetic acid twisting method test
Get 96 of healthy mices, ♀ ♂ half and half is divided into distilled water group, benorylate 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 (abdominal part shrinks indent, trunk and back leg extension, hips up) of respectively organizing mice in the injection glacial acetic acid 15min and writhing response occurring immediately.Organize the analgesia suppression ratio (table 44) that a t checked and be calculated as follows the administration group.
Table 44 sulphuring and sulphuring Radix Angelicae Dahuricae sample alcohol extract mice acetic acid twisting method repeated trials one result not
(
n＝12)
Compare with the distilled water group,
*P＜0.05,
*P＜0.01,
* *P＜0.001
1.3.3 mice acetic acid twisting method repeated trials two is got 96 of healthy mices, ♀ ♂ half and half is divided into distilled water group, benorylate group (20gkg at random
-1) 2 groups, No. 408 (No. 435) (crude drug 2.5,5,10gkg
-1) 6 groups.Totally 8 groups.Route of administration, experimental technique are with under " 1.3.1 " item (table 45).
Table 45 sulphuring and sulphuring Radix Angelicae Dahuricae sample alcohol extract mice acetic acid twisting method repeated trials two results not
(
n＝12)
Compare with the distilled water group,
*P＜0.05,
* *P＜0.001
2 discuss
2.1 by adopting mice acetic acid twisting method to sulphuring and the not comparison of sulphuring angelica root analgesic activity, the result shows, benorylate 20gkg
-1Group demonstrates significant analgesia role (P＜0.001); Average writhing response number of times of mice and distilled water matched group are relatively after each sample alcohol extract administration of the Radix Angelicae Dahuricae, each dosage group of three samples of sulphuring does not demonstrate analgesic activity (P＞0.05), and under same experimental condition, four dosage 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) (P＜0.05) can obviously reduce the mouse writhing number of times due to the glacial acetic acid, and significant difference is all arranged, and demonstrates tangible analgesic effect.
2.2 lot of experiments result shows, sulphuring all has a significant impact the chemical constituent and the analgesic activity of angelica root, some countries carry out the limit detection of sulfur to the Chinese crude drug of China's outlet in addition, the sulphuring habitat processing method of Cai Yonging is demanded urgently improving now, the standardized study of Radix Angelicae Dahuricae processing in production place is strengthened in suggestion, adopt suning method or science, convenient and practical processing method more, to guarantee quality of medicinal material and drug safety.
To sum up, adopt mice acetic acid twisting method to observe mice 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 matched group relatively, the sample sets there was no significant difference of sulphuring (P＞0.05), 3 of sulphuring Radix Angelicae Dahuricae samples do not cause the writhing response number of times that pain causes to the mice 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 medical material does not then demonstrate significant analgesia role.Wherein the content difference with oxypeucedanin is bigger, and this experiment showed, the quality by the controlled oxidation peucedanin, can reach the purpose of the quality of control Radix Angelicae Dahuricae preparation.
Claims (12)
1. Radix Angelicae Dahuricae extract, it is characterized in that: it contains oxypeucedanin 9.50-14.24mg/g.
2. Radix Angelicae Dahuricae extract according to claim 1 is characterized in that: it also contains imperatorin 5.52-8.28mg/g.
3. Radix Angelicae Dahuricae extract according to claim 1 and 2 is characterized in that: it is the water or the extractive with organic solvent of angelica root.
4. Radix Angelicae Dahuricae extract according to claim 3 is characterized in that: it is to be prepared from by following method: get the angelica root coarse powder, add the 50-95% ethanol extraction; Extracting solution promptly gets Radix Angelicae Dahuricae extract through 60-70 ℃ of concentrating under reduced pressure drying.
5. Radix Angelicae Dahuricae extract according to claim 4 is characterized in that: described ethanol extraction method is percolation or ultrasonic extraction.
6. according to any described Radix Angelicae Dahuricae extract of claim 1-5, it is characterized in that: the HPLC finger printing of described Radix Angelicae Dahuricae extract as shown in Figure 2, chromatographic condition is: chromatographic column: Diamonsil
TMC
18HPLC chromatographic column (250 * 4.6mm, 5 μ m); Mobile phase: methanol-oxolane-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.
7. pharmaceutical composition, it is to be active component to contain each described Radix Angelicae Dahuricae extract of claim 1-6, adds the medicament that acceptable accessories or complementary composition are prepared from.
8. pharmaceutical composition according to claim 7 is characterized in that: described medicament is capsule, tablet, granule, pill, oral liquid, soft capsule or drop pill.
9. method for quality that detects angelica root, the described Radix Angelicae Dahuricae extract of claim 1 or contain the pharmaceutical composition of Radix Angelicae Dahuricae 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, by high effective liquid chromatography for measuring, the ultraviolet detection wavelength is 305nm;
B, get oxypeucedanin, imperatorin reference substance, be prepared into reference substance solution;
The preparation of c, need testing solution: get angelica root, extract or preparation, add methanol, power 300W, the supersound extraction 0.5-1.5 of frequency 50kHz hour, take out, put coldly, be diluted to scale with methanol, shake up, filter, get subsequent filtrate, promptly;
D, accurate reference substance solution and the need testing solution injection chromatograph of liquid drawn of difference are measured, promptly.
10. quality determining method according to claim 9 is characterized in that: the described chromatographic condition of a step is: chromatographic column: Diamonsil
TMC
18HPLC chromatographic column (250 * 4.6mm, 5 μ m); Mobile phase: methanol-oxolane-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.
11. according to claim 9 or 10 described quality determining methods, it is characterized in that: the content of oxypeucedanin must not be lower than 0.12%w/w in the described angelica root, the content of imperatorin must not be lower than 0.1%w/w.
12. pharmaceutical composition according to claim 11 is characterized in that: contain oxypeucedanin 0.12-0.23%w/w, imperatorin 0.11-0.22%w/w in the described angelica root.
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Non-Patent Citations (1)
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| 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 20090715 高颖 白芷配方颗粒质量标准的规范化研究 E057-277 1-11 , 第7期 * |
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