CN101637486B - Angelica dahurica coumarin extract as well as preparation method and quality control method thereof - Google Patents
Angelica dahurica coumarin extract as well as preparation method and quality control method thereof Download PDFInfo
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Abstract
The invention provides an angelica dahurica coumarin extract, wherein the total content of coumarin is not lower than 44.672mg/g, and the content of imperatorin is not lower than 68.623mg/g. The invention further provides an extraction method and a quality control method of the extract. The angelica dahurica coumarin extract has high extraction purity, high content of coumatin, stable quality and function of 5-hydroxytryptamine regulation, is capable of treating diseases caused by 5-hydroxytryptamine besides migraine, has distinguished efficacy and strong pertinency of indications, and provides a new choice for clinical treatment.
Description
Technical field
The present invention relates to angelica dahurica coumarin extract and method of quality control, belong to drug world.
Background technology
The Radix Angelicae Dahuricae is a kind of conventional Chinese medicine, has the effect of wind dispelling cold expelling, dampness evacuation of pus, is usually used in treating diseases such as anemofrigid cold, nasosinusitis headache." Radix Angelicae Dahuricae that records of Chinese pharmacopoeia version in 2000 is the dry root of samphire Radix Angelicae Dahuricae Angelica dahurica (Fisch.ex Hoffm.) Benth.et Hook.f. or Radix angelicae dahuricae A.dahurica (Fisch.ex Hoffm.) Benth.etHook.f.var.formosana (Boiss.) Shan et Yuan.Contained effective ingredient is mainly coumarin kind compounds such as oxypeucedanin, imperatorin, isoimperatorin.Angelica dahurica coumarin has antiinflammatory, suppresses the generation of PGE2, antihistamine effect, photosensitization, to the effect of adipose cell, the effect of smooth muscle, anti-microbial effect etc. (are seen: Du Xingxu etc., the progress of Chinese medicine angelica dahurica coumarin constituents, Jinggangshan College's journal (natural science), the 26th the 2nd phase of volume, in October, 2005); Qin Xuhua etc., the angelica anomala total coumarins is treated migrainous part pharmacodynamic experiment research, the Sichuan traditional Chinese medical science, 2008 06 phases, report Chinese medicine angelica dahurica coumarin causes experimental migrainous drug effect to nitroglycerin.Still there be not at present the bibliographical information of relevant angelica dahurica coumarin extract as the medicine of 5-hydroxy tryptamine regulator.
Existing pertinent literature report extracts the Coumarins composition from the Chinese medicine Radix Angelicae Dahuricae, from the ether of the Radix Angelicae Dahuricae and ethanol extraction, get stearic acid as: utilization silica gel column chromatography methods such as Zhang Hanqing, bergapton, the aqua oxidation peucedanin, imperatorin, isoimperatorin, other imperatorin (alloimperatorin), byak-angelicin, coumarin compositions such as oxypeucedanin (Zhang Hanqing, Yuan Changqi, Chen Guiying, Deng. the The Chemical Constituents of Radix angelicae dahuricae root [J]. the pharmacy circular, 1980,15k:386.), wherein the document spininess is to the report of particular compound, glad as Huang, Su Lequn, Shao Wei, Deng, the component analysis of angelica dahurica coumarin chemical compound and CO
2The research of supercritical extraction process. China Dispensary, 2005,16 (11): 824～825.And at the extracting method of angelica dahurica coumarin, reported that at present the employing percolation extracts, as: Yuan Yu, percolation extract the technical study of total coumarin angelica dahurica, the time 2008 the 19th the 2nd phases of volume of precious traditional Chinese medical science traditional Chinese medicines.Reported in the document and adopted 95% ethanol to carry out ultrasound wave, hot reflux extraction, percolation extraction, it is higher that wherein percolation has obtained total coumarins content, but the total coumarin extract that obtains only extracts by thick, without purifies and separates, the content of the total coumarins that obtains is low, can not reach medicinal requirement.
In addition, the method for quality control of the Radix Angelicae Dahuricae of report is more at present, as: Zhang Xiuqin, Xu Li lamp stand. the polarographic method of effective ingredient in the Chinese medicine Radix Angelicae Dahuricae. pharmaceutical analysis magazine, 1983,3 (4): 218; Li Hongyu wears and makes a leapleap forward, Xie Chengke, etc. the rp-hplc analysis of Coumarins composition in the angelica anomala. West China pharmaceutical journal, 1990,5 (4): 231; Wang Li people, Li Hongyu, Xie Chengke. the rp-hplc of Coumarins composition in the Radix Angelicae Dahuricae. Acta Pharmaceutica Sinica, 1990,25 (2): 131, be to adopt product imperatorin, the index components in the total coumarinss such as isoimperatorin, oxypeucedanin, the ultraviolet maximum absorption wavelength performance of imperatorin, isoimperatorin, oxypeucedanin is different, but all has the absorption band of broad about 254nm; Or measure with content total fragrant plain in the Radix Angelicae Dahuricae.Because the complicated component that contains in the natural drug live part only passes through wherein several index components, can not accurately judge the quality of effective site.
Summary of the invention
Technical scheme of the present invention has provided a kind of angelica dahurica coumarin extract.Another technical scheme of the present invention has provided the method for quality control of this extract.
The invention provides angelica dahurica coumarin extract, wherein, the content of total coumarins should be not less than 44.672mg/g, and the content of imperatorin should be not less than 68.623mg/g.
The HPLC chromatograph of extract of the present invention contains 14 total fingerprint peakses as shown in Figure 1;
Its chromatographic condition is:
Chromatographic column: Aichrom Reliasil C
18Chromatographic column (4.6mm * 250mm, 5 μ m), mobile phase: methanol, water and oxolane gradient elution, column temperature: 40 ℃, flow velocity: 1ml/min, wavelength: 300nm, sample size: 5 μ l, methanol, water and oxolane gradient elution time and ratio are:
| Elution time (min) | A water (%) | B oxolane (%) | C methanol (%) |
| 0 | 60 | 8 | 32 |
| 10 | 60 | 8 | 32 |
| 50 | 20 | 16 | 64 |
Wherein, the average relative retention time (min) at described total peak is: 7.911,9.731,13.390,16.59,19.997,23.964,29.512,30.271,31.949,34.525,37.298,39.161,39.957,42.096.
The present invention also provides a kind of method for quality of controlling angelica dahurica coumarin extract, and it is to adopt the HPLC finger printing to carry out quality control, and wherein, chromatographic condition is:
Chromatographic column: Aichrom Reliasil C
18Chromatographic column (4.6mm * 250mm, 5 μ m), mobile phase: methanol, water and oxolane gradient elution, column temperature: 40 ℃, flow velocity: 1ml/min, wavelength: 300nm, sample size: 5 μ l, methanol, water and oxolane gradient elution time and ratio are:
| Elution time (min) | A water (%) | B oxolane (%) | C methanol (%) |
| 0 10 50 | 60 60 20 | 8 8 16 | 32 32 64 |
The present invention also provides a kind of method for preparing described angelica dahurica coumarin extract, comprises the steps:
A, Radix Angelicae Dahuricae 55-95% ethanol extract decompression recycling ethanol concentrate;
B, a step is concentrated adds petroleum ether extraction, discards the petroleum ether extraction layer; Raffinate phase adds ethyl acetate extraction, merges extraction phase, the reclaim under reduced pressure ethyl acetate, promptly.
Wherein, the described angelica root granularity of a step is: 20 orders, ethanol extraction are that 75% alcohol heat reflux extracts.
Angelica dahurica coumarin extract of the present invention, the dna purity height, the content height of total coumarins, steady quality has the 5-hydroxy tryptamine regulating action, except the treatment migraine, also can treat 5-hydroxy tryptamine and reduce other disease that causes, drug effect is clear and definite, and indication is with strong points, provides a kind of new selection for clinical.
Obviously, according to foregoing of the present invention,,, can also make modification, replacement or the change of other various ways not breaking away under the above-mentioned basic fundamental thought of the present invention prerequisite according to the ordinary skill knowledge and the customary means of this area.
Description of drawings
Fig. 1 marks the sample HPLC chromatogram at total peak
The HPLC finger printing of Figure 21 0 batch sample
Fig. 3 Radix Angelicae Dahuricae granularity is investigated the result
The specific embodiment
The preparation of embodiment 1 angelica dahurica coumarin of the present invention
Take by weighing angelica root coarse powder (20 order) 5000g, totally 10 parts, add 8 times of amount alcohol reflux of 75% respectively 3 times, each 3 hours, filter merge extractive liquid,, decompression recycling ethanol, it is that every ml is equivalent to the 2g medical material that medicinal liquid is evaporated to concentration, put cold, add 2 times of amount petroleum ether extractiones 1 time respectively after, discard petroleum ether layer (being extraction phase), raffinate phase adds ethyl acetate extraction respectively 3 times again, each 2 times of amount ethyl acetate, merging extraction phase, reclaim under reduced pressure ethyl acetate of adding, extractum is carried out the vacuum concentration drying, promptly get the angelica anomala coumarin extract.
Because total coumarin angelica dahurica is big class principal component and the mixture formed of principal component not, directly pass through the physicochemical property of Coumarins composition, be very difficult from plant, extract content height, purity height, stay-in-grade total coumarins.The experimental basis that each parameter is selected in following examples explanation extraction process.
The extraction process screening test of embodiment 2 angelica anomala coumarins
1, instrument and material
1.1 instrument
752 type ultraviolet spectrometry degree meters (Shanghai Spectrum Apparatus Co., Ltd.); IS09001 electronic balance (Beijing Sai Duolisi balance company limited); KQ-100DE type numerical control supersonic washer (Kunshan Ultrasonic Instruments Co., Ltd.); HA121-50-01 type supercritical CO
2Extraction equipment (Huaan, Jiangsu supercritical extraction company limited); RE-52A Rotary Evaporators (Shanghai Yarong Biochemical Instrument Plant); The circulating vacuum pump of SHB-3 (Zhengzhou Tu Fu instrument plant); HH-S digital display thermostat water bath (Jintan City, Jiangsu Province Medical Instruments factory); The SL500 steel percolation jar (Hengyang City Jinshan Traditional Chinese Medicine Equipment Manufacturing Co., Ltd) that becomes rusty; The DHG-9070A electric heating warm air dry oven (Shanghai Qi Xin Instr Ltd.) that weighs.
1.2 material
Methanol, ethanol are analytical pure (Chengdu Ke Long chemical reagent work); 95% medical alcohol; The imperatorin reference substance (is provided by Nat'l Pharmaceutical ﹠ Biological Products Control Institute, lot number: 110826-200511), angelica anomala is accredited as the dry root of samphire angelica anomala Angelica dahurica (Fisch.ex Hoffm.) Benth.et Hook.f. available from Suining, Mianyang City, Langzhong City Chinese crude drug company through professor Ma Yuying of TCD identification teaching and research room of Chengdu University of Traditional Chinese Medicine..
2, experimental technique and result
2.1 determining of the medical material place of production
The Radix Angelicae Dahuricae is the genuine medicinal materials in Sichuan, in recent years, Study on Technology has all been carried out Radix Angelicae Dahuricae growth, fostered and has been introduced a fine variety in many inside the province areas, caused the uneven present situation of angelica root quality, for stability and the homogeneity that guarantees end product quality, the content of the total coumarins that the angelica anomala in the different places of production is inside the province extracted is measured, to determine the best place of production.
The different place of production of table 1 angelica anomala total coumarins assay result
According to the said determination result, total coumarins content is relatively higher than the angelica anomala medical material of Mianyang and Langzhong in the angelica anomala medical material of the place of production, Suining, so select the Suining angelica anomala as the experimental drug material.
2.2 the comparative test of different angelica anomala coumarin extracting method
This experiment is an evaluation index with total coumarins content, to extracting method (ultrasonic extraction, heat reflow method, the percolation, and supercritical CO of the Radix Angelicae Dahuricae
2Extraction) carries out system, relatively comprehensive, thereby determine the optimum extracting method of angelica anomala total coumarins.
(1) experimental technique
1. ultrasonic extraction
Get Radix Angelicae Dahuricae coarse powder (crossing 20 mesh sieves) 500g, add 8 times of amount 75% ethanol, soaked 1 hour, supersound extraction 3 hours (ultrasound condition: power 500W, frequency 50KHz) is collected extracting solution, measures total fragrant cellulose content.
2. heat reflow method
Get Radix Angelicae Dahuricae coarse powder (crossing 20 mesh sieves) 500g, add 8 times of amount 75% ethanol, hot reflux was extracted 3 hours, collected extracting solution, measured total fragrant cellulose content.
3. percolation
Get Radix Angelicae Dahuricae coarse powder (crossing 20 mesh sieves) 500g, add 8 times of amount 75% ethanol,, collect percolate, measure total fragrant cellulose content with the speed percolation of 1ml/min/Kg.
4. supercritical CO
2Extraction
Get Radix Angelicae Dahuricae coarse powder (crossing 20 mesh sieves) 500g, add in the supercritical extraction unit, carry out supercritical CO
2Extraction (extraction conditions: 50 ℃ of temperature, pressure 30MPa, extraction 3.5h, separating pressure 7.0MPa extracted 3 hours) and collect extract, measure total coumarins content
(2) experimental result
Extracting solution total coumarins content to the Different Extraction Method gained compares.The results are shown in Table 2.
The comparison of table 2 Different Extraction Method
| Extracting method | Total coumarins content (mg/g) |
| Ultrasonic extraction | 6.480 |
| Hot reflux is extracted | 10.971 |
| Percolation extracts | 9.020 |
| The |
7.514 |
As seen from the above table, by the comparison of four kinds of methods, it is the highest that the result extracts institute's total coumarin angelica dahurica content of being surveyed with heat reflow method, and this method is simple to operate, is applicable to big production research, therefore, selects the heat reflow method extraction for use.
2.3 the angelica anomala alcohol reflux is inhaled the investigation of pure rate
Take by weighing angelica anomala coarse powder 100g, add the 500ml75% soak with ethanol, observe the situation of medical material moistening, treat that medical material all soaks into, leach whole unabsorbed solvents, calculate and inhale pure rate, the results are shown in Table 3.
Table 3 angelica anomala inhales pure rate and investigates the result
Above result shows: it is 109.3% that the angelica anomala medical material is inhaled pure rate, so when the angelica anomala alcohol reflux, medical material is added the ethanol of definite concentration of 109.3% for the first time.
2.4 the investigation of angelica anomala reflux, extract, medical material granularity
For the extraction effect of investigating angelica anomala medical material total coumarins and the relation of granularity, respectively four kinds of medical material granularities (10 orders, 20 orders, 30 orders and 40 orders) are investigated.
(1) experimental technique
Take by weighing each 100g of medical material of four kinds of granularities (10 orders, 20 orders, 30 orders and 40 orders), add the ethanol of 8 times of amounts 75%, reflux, extract, 3 times, each 1h collects extracting solution, measures the content of total coumarins.
(2) experimental result
Different medical material granularity gained extracting solution total coumarins content are compared, and it the results are shown in Table 4, Fig. 3.
Table 4 medical material granularity is investigated the result
| Medical material granularity (order) | Total coumarins content (mg/g) |
| 10 | 8.593 |
| 20 | 9.657 |
| 30 | 9.564 |
| 40 | 9.235 |
The above results shows: when the medical material granularity was 20 orders, the total coumarins extraction ratio was higher relatively in the medical material, and therefore, adopting granularity is that 20 purpose medical materials extract.
2.5 angelica anomala reflux, extract, technical study
(1) experimental technique
1. orthogonal test factor level design
Adopt the alcohol heat reflux method to extract, select concentration of alcohol, ethanol consumption, extraction time and extraction time four major influence factors, each factor is selected three levels (seeing Table 5), presses L
9(3
4) orthogonal table arrangement test (seeing Table 6).Angelica root powder (20 order) 20g is got in each test, and amount to 9 parts and test, be evaluation index with total coumarin angelica dahurica content and dried cream rate, carry out comprehensive grading, its proportion is respectively 80%, 20%, therefrom screens best ethanol extraction technology.
Table 5 factor level table
| Factor | Concentration of alcohol (%) A | Ethanol consumption (doubly) B | Extraction time (inferior) C | Extraction time (h) D |
| 1 | 55 | 4 times | 1 | 1 |
| 2 | 75 | 6 |
2 | 2 |
| 3 | 95 | 8 |
3 | 3 |
2. the preparation of need testing solution
Take by weighing angelica root (20 order) 20g, test according to orthogonal test table, extracting solution is settled to 100～500ml, draw 5ml respectively in the evaporating dish of constant weight, water bath method is with dissolve with methanol and be settled to 50ml, shake up, accurate respectively absorption 0.8～2ml puts in the 10ml measuring bottle, is diluted to scale with methanol, shakes up, as need testing solution, with methanol is blank, measures absorbance in the 300nm place, and calculates total coumarin angelica dahurica content according to standard curve.
(2) experimental result
Traditional Chinese medical science system software kit SPSS carried out Treatment Analysis during orthogonal experiments adopted, and the results are shown in Table 6 and table 7.
Table 6 Radix Angelicae Dahuricae alcohol extraction orthogonal experiment plan is taken into account the result
Annotate: Z=(y
1/ y
Max) * 20+ (y
2/ y
Max) * 80
Table 7 analysis of variance table
Annotate: F
(0.01 2,2)=99 F
(0.05 2,2)=19.0 F
(0.1 2,2)=9
From The results of analysis of variance as can be seen: influence factor's primary and secondary of angelica anomala alcohol reflux is in proper order: C＞D＞A＞B.The C factor influence significance (P＜0.05), optimum condition is: A
2B
3C
3D
3, promptly add 8 times and measure 75% ethanol, extract 3 times, extract 3h at every turn.
2.6 angelica anomala reflux, extract, process certification test
(1) experimental technique
For science, reasonability and the feasibility of verifying The above results, take by weighing 3 parts of angelica anomala medical materials, each 500g carries out repeated trials by above definite optimum extraction condition.
(2) experimental result
Demonstration test the results are shown in Table 8.
Table 8 demonstration test result
From above-mentioned demonstration test result as can be known: the technology that is filtered out is rationally feasible, and operability and repeatability are better.
The conditional parameter of embodiment 3 angelica anomala coumarin separation purifying techniques is selected test
1 instrument and material
1.1 instrument
752 type ultraviolet spectrometry degree meters (Shanghai Spectrum Apparatus Co., Ltd.); RE-52A Rotary Evaporators (Shanghai Yarong Biochemical Instrument Plant); The circulating vacuum pump of SHB-3 (Zhengzhou Tu Fu instrument plant); HH-S digital display thermostat water bath (Jintan City, Jiangsu Province Medical Instruments factory); ISO9001 electronic balance (Beijing Sai Duolisi balance company limited); Centrifuge refrigerated centrifuger (German Eppendorf company); PHs-2c type acidometer (Chengdu Noah's ark scientific and technological development company)
1.2 material
Chloroform, cyclohexane extraction, ether, ethyl acetate, petroleum ether, hydrochloric acid, sodium chloride, carbonic acid chlorine sodium and p chloromethylbenzoic acid sodium are analytical pure (Chengdu Ke Long chemical reagent work)
2, experimental technique and result
2.1 sample liquid preparation
Angelica anomala reflux, extract, technology according to embodiment 2, take by weighing 5kg angelica anomala medical material, the ethanol that adds 8 times of amounts 75%, extract 3 times, each 3h, merge extractive liquid,, (concentration that is medicinal liquid is that every ml medicinal liquid is equivalent to the 2g medical material to decompression recycling ethanol to 2500ml, the concentration of following medicinal liquid is expressed as 2g/ml), standby.
2.2 the angelica dahurica coumarin isolation and purification method is investigated
Physicochemical property and construction features according to the angelica anomala coumarin, selected acid precipitation, alkali extraction and acid precipitation, macroporous resin separation and organic solvent extraction several method that Radix Angelicae Dahuricae ethanol extract is carried out separation and purification, and the principal element that influences various purification process investigated, content and retention rate with the angelica anomala total coumarins are evaluation index, its purifying process is optimized screening optimal separation purification process.
(1) acid precipitation partition method
1. pH value is investigated
Get above-mentioned sample liquid (2g/ml) 20ml, totally 7 parts, transfer to different pH value respectively, put in the refrigerator to place and spend the night, centrifugal (4000r/min) 15 minutes measures in the precipitation total coumarins content and calculate its retention rate, thereby the screening optimal pH the results are shown in Table 9.
Table 9pH value is investigated the result
| PH value | Coumarin content (%) in the precipitation | Coumarin retention rate (%) in the precipitation |
| 1.0 | 20.02 | 70.89 |
| 2.0 | 23.88 | 71.25 |
| 2.5 | 23.96 | 75.29 |
| 3.0 | 23.98 | 76.48 |
| 3.5 | 22.02 | 72.25 |
| 4.0 | 22.12 | 69.57 |
| 4.5 | 21.83 | 68.45 |
| Material liquid | 6.82 | 100 |
The above results shows: the preferable pH value of acid precipitation effect is 2～3, and in this pH value scope, coumarin content and retention rate are all higher, and therefore the pH value with acid precipitation is decided to be 2～3.
2. temperature is investigated
The pH of above-mentioned sample liquid (2g/ml) is adjusted to 2～3, gets 20ml, totally 6 parts, spend the night respectively at 0 ℃, 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃ placements, centrifugal (4000r/min) 15 minutes measures in the precipitation total coumarins content and calculates the total coumarins retention rate, and it the results are shown in Table 10.
Table 10 temperature is investigated the result
| Temperature (℃) | Coumarin content (%) in the precipitation | Coumarin retention rate (%) in the |
| 0 | 20.02 | 71.25 |
| 10 | 22.88 | 70.68 |
| 20 | 23.17 | 75.88 |
| 30 | 22.93 | 76.32 |
| 40 | 21.59 | 72.91 |
| 50 | 20.02 | 69.83 |
| Material liquid | 6.82 | 100 |
The above results shows: dwell temperature in 0～50 ℃ of scope, there was no significant difference, but dwell temperature 20 ℃～30 ℃ preferable relatively, approaching with room temperature, so dwell temperature is decided to be room temperature.
But: when adopting the acid precipitation method to handle, the content of total coumarins is lower in the precipitation, and retention rate is also lower, finds that simultaneously the contained total fragrant element of supernatant is more, fails to reach re-set target, therefore, considers to adopt other method to carry out purification
(2) alkali extraction and acid precipitation method
This method is one of classical partition method of coumarin, be because coumarin has phenolic hydroxyl group or lactone structure, can in aqueous slkali, dissociate or open loop generation soluble-salt, add and revert to lactone after the acid reduction and be insoluble to sour water, and impurity does not dissolve in alkaline environment or alkalescence and sour environment in all dissolve, so can utilize the alkali extraction and acid precipitation method that it is separated.
1. the kind of alkali and concentration are investigated
Get above-mentioned sample liquid (2g/ml) 20ml, totally 8 parts, add following alkali liquor 100ml respectively, hot reflux 1 hour is put coldly, filters, and measures the content of total coumarins in the filtrate and calculates the retention rate of total coumarins, and the kind and the concentration of screening alkali the results are shown in Table 11.
The kind of table 11 alkali and concentration are investigated the result
| The alkali kind | Total coumarins content (%) in the filtrate | Total coumarins retention rate (%) in the filtrate |
| 0.5％NaOH | 11.32 | 85.13 |
| 1％NaOH | 3.01 | 44.84 |
| 0.1％NaOH | 7.31 | 67.18 |
| 0.5％Ca(OH) 2 | 2.31 | 31.42 |
| 5％Na2CO3 | 5.01 | 49.34 |
| 5％Na2HCO3 | 4.68 | 45.42 |
| 5％Na2HPO3 | 4.86 | 47.28 |
| 0.5％KOH | 10.02 | 83.37 |
| Material liquid | 6.82 | 100 |
The above results shows: when adopting 0.5%NaOH solution to dissolve, total coumarins content and retention rate are higher relatively in the filtrate, and it is molten that therefore definite employing 0.5%NaOH carries out alkali.
2. Radix Angelicae Dahuricae ethanol extract alkali extraction and acid precipitation technology is investigated:
Get above-mentioned sample liquid (2g/ml) 20ml, totally 3 parts, add 100ml0.5%NaOH solution respectively, reflux 1h is after the cooling, carrying out acid precipitation by the best purifying process condition of acid precipitation (is adjust pH 2～3, the room temperature standing over night, centrifugal (4000r/min) 15 minutes gets the precipitation part, measure the content of total coumarins in the precipitation and calculate the retention rate of total coumarins, the results are shown in Table 12.
Table 12 Radix Angelicae Dahuricae ethanol extract alkali extraction and acid precipitation technology table
| Test number (TN) | Total coumarins content (%) in the precipitation | Total coumarins retention rate (%) in the precipitation |
| 1 | 24.81 | 74.72 |
| 2 | 26.54 | 79.59 |
| 3 | 26.97 | 80.63 |
| Material liquid | 6.82 | 100 |
[0128]The above results shows: alkali extraction and acid precipitation technology is more stable, but medicinal liquid precipitates less behind acid precipitation.Total fragrant cellulose content and retention rate are relatively low in the precipitation, and be little with acid precipitation processing difference.
Alkali concn is too high or alkaline when strong in the molten process of alkali, the total coumarins retention rate is lower, this may be owing to long-time heating in strong alkali environment, the lactone constituent structure is destroyed, fail reduction after the acidify, still existing in solution with the form of salt causes retention rate low, adopts the alkali extraction and acid precipitation method to handle simultaneously, to the coumarin of soda acid sensitivity, possibly can't obtain the former material of depositing.Therefore, consider to adopt other method to carry out purification.
(3) macroreticular resin absorbing method
1. the investigation of macroporous resin kind
It is an amount of to draw above-mentioned sample liquid (2g/ml), being diluted to concentration is the medicinal liquid that every ml is equivalent to the 0.5g medical material, get 20ml, totally 5 parts, the macroporous adsorptive resins that is added on different model respectively pushes up, and first water is eluted to colourless, colourless with 75% ethanol elution again to effluent, collect 75% ethanol elution, measure total coumarins content in the eluent and calculate the retention rate of total coumarins, the results are shown in Table 13.
Table 13 macroporous resin kind is investigated the result
| Tested number | The resin model | Eluent coumarin content (%) | Coumarin retention rate (%) |
| 1 | D101 | 16.23 | 50.14 |
| 2 | SP70 | 15.35 | 42.86 |
| 3 | AB-8 | 29.83 | 65.28 |
| 4 | D2820 | 17.94 | 52.11 |
| 5 | X-5 | 14.52 | 40.12 |
| Original liquid | ? | 6.82 | 100 |
The above results shows: AB-8 macroporous resin separation and purification effect is better, and total coumarins content improves a lot in the eluent, for further improving the relative amount and the retention rate of total coumarins effective site, intends the other factors of influence absorption is investigated.
2. the investigation of eluting solvent
It is an amount of to draw above-mentioned sample liquid (2g/ml), being diluted to concentration is the medicinal liquid that every ml is equivalent to the 0.5g medical material, get 20ml, totally 4 parts, be added on 80mlAB-8 macroporous resin capital respectively, first water is eluted to colourless, colourless with 45% ethanol, 60% ethanol, 75% ethanol, 90% ethanol elution respectively again to effluent, collect ethanol elution, measure eluent total coumarins content and calculate the retention rate of total coumarins, it the results are shown in Table 14.
Table 14 eluting solvent is investigated the result
| Tested number | The eluting solvent | Eluent total coumarins content (%) | Coumarin retention rate (%) |
| 1 | 45% ethanol | 8.63 | 28.31 |
| 2 | 60% ethanol | 17.13 | 46.18 |
| 3 | 75% ethanol | 29.67 | 64.25 |
| 4 | 90% ethanol | 30.42 | 65.92 |
| Original liquid | ? | 6.82 | 100 |
[0140]Above result shows: with 45% ethanol as eluting solvent, the coumarin elute effect is relatively poor, most of coumarin is not by desorbing, with 75%, 90% ethanol as eluting solvent, the coumarin elute effect is better but retention rate is still lower, and test shows, uses the macroporous adsorbent resin remove impurity, the active substance loss is bigger, so consider to adopt other impurity-removing method to improve the content and the retention rate of coumarin effective site in the extractum.
(4) organic solvent extractionprocess
The angelica anomala coumarin is fat-soluble stronger, be soluble in organic solvents such as chloroform, cyclohexane extraction, ether, ethyl acetate, petroleum ether, so consider to adopt above organic solvent extraction to carry out the separation and purification of medicinal liquid, be index with angelica anomala total coumarins content, screen optimum extraction process.
1. the screening of organic solvent kind
Get above-mentioned sample liquid (2g/ml) 20ml, totally 5 parts, add chloroform, cyclohexane extraction, ethyl acetate, ether and petroleum ether respectively and extract, extract 3 times, add 40ml, combining extraction liquid at every turn, measure in the extraction phase total coumarins content and calculate the total coumarins retention rate, the results are shown in Table 15.
Table 15 organic solvent extraction result of the test
| Solvent species | Total coumarins content (%) in the extraction phase | Total coumarins retention rate (%) in the extraction phase |
| Chloroform | 26.57 | 81.98 |
| Cyclohexane extraction | 9.85 | 13.28 |
| Ethyl acetate | 33.42 | 92.37 |
| Ether | 30.24 | 84.32 |
| Petroleum ether | 9.45 | 12.04 |
| Material liquid | 6.82 | 100 |
The above results shows: adopt ethyl acetate extraction, total coumarins content and retention rate are than other organic solvent height.
Find in the experimentation to adopt above-mentioned organic solvent to extract, the oils and fats sexual element is more in the gained extract, so that total coumarins content is on the low side, from last table result as can be seen, petroleum ether is not high to the extraction yield of angelica anomala Coumarins composition, and that petroleum ether takes off the ester effect is better, therefore, the employing petroleum ether takes off ester earlier to be handled, and re-uses in the table the higher chloroform of total coumarins extraction yield, ethyl acetate and the ether raffinate phase to petroleum ether is extracted.
2. with the screening of the organic solvent of petroleum ether coupling
Get above-mentioned sample liquid (2g/ml) 20ml, totally 3 parts, add the 40ml petroleum ether extraction respectively 1 time, discard petroleum ether layer (being extraction phase), raffinate phase adds chloroform, ethyl acetate and ether more respectively and extracts, and extracts respectively 3 times, respectively add 40ml at every turn, measure angelica anomala total coumarins content in chloroform, ethyl acetate and the ether extraction liquid respectively and calculate the retention rate of total coumarins, thereby determine and petroleum ether coupling effect of extracting preferred organic solvents, the results are shown in Table 16.
Table 16 is investigated the result with petroleum ether coupling organic solvent
| Solvent species | Total coumarins content (%) in the extraction phase | Total coumarins retention rate (%) in the extraction phase |
| Chloroform | 43.87 | 71.11 |
| Ethyl acetate | 59.78 | 82.26 |
| Ether | 47.52 | 74.73 |
| Material liquid | 6.82 | 100 |
The above results shows: extracting solution behind petroleum ether extraction, raffinate phase reuse ethyl acetate extraction, the total coumarins content that obtains is higher relatively, simultaneously ethyl acetate is safe in chloroform and ether.Therefore, select for use ethyl acetate as with the organic solvent of petroleum ether coupling.
3. petroleum ether-ethyl acetate coupling extraction process
In order to determine the optimum process condition of separation and purification, the major influence factors in the petroleum ether-ethyl acetate coupling extracting process is investigated.
The investigation of A petroleum ether extraction number of times
Get above-mentioned sample liquid (2g/ml) 20ml, totally 3 parts, use petroleum ether extraction respectively 1 time, 2 times, 3 times, each petroleum ether 40ml that adds, measure total coumarins content in the raffinate phase of different extraction times respectively and calculate the retention rate of total coumarins, to determine the petroleum ether extraction number of times, it the results are shown in Table 17.
Table 17 petroleum ether extraction number of times is investigated the result
| Extraction times (inferior) | Total coumarins content (%) in the raffinate phase | The retention rate (%) of total legumin in the raffinate phase |
| 1 | 12.78 | 90.08 |
| 2 | 10.33 | 89.47 |
| 3 | 9.87 | 87.15 |
| Material liquid | 6.82 | 100 |
The above results shows: along with the increase of petroleum ether extraction number of times, the content of total coumarins slightly reduces in the raffinate phase, this may be because along with the petroleum ether consumption increases, the removed while of lubricant component, the loss of Coumarins composition increases, therefore, determine that according to last table result the petroleum ether extraction number of times is 1 time.
The investigation of B petroleum ether consumption
Get above-mentioned sample liquid (2g/ml) 20ml, totally 3 parts, add 20ml, 40ml, 60ml (i.e. 1 times of amount, 2 times of amounts, 3 times of amounts) petroleum ether extraction respectively 1 time, discard extraction phase, measure total coumarins content in the raffinate phase and calculate the retention rate of total coumarins, to determine the consumption of petroleum ether, it the results are shown in Table 18.
Table 18 petroleum ether consumption is investigated the result
| Petroleum ether consumption (doubly) | Total coumarins content (%) in the raffinate phase | The retention rate (%) of total legumin in the raffinate phase |
| 1 | 10.95 | 91.08 |
| 2 | 12.63 | 90.28 |
| 3 | 12.97 | 89.35 |
| Material liquid | 6.82 | 100 |
The above results shows: the petroleum ether consumption is 2 times of amounts when above, and the increase of total coumarins content is more obvious in the raffinate phase.But along with the petroleum ether consumption continues to increase, the coumarin changes of contents is little, therefore, considers from saving the cost aspect, adopts 2 times of amount petroleum ether to extract.
The investigation of C ethyl acetate consumption
Get totally 3 parts of above-mentioned sample liquid (2g/ml), each 20ml, add petroleum ether 40ml extraction 1 time respectively, discard extraction phase, raffinate phase adds 20ml, 40ml, 60ml (i.e. 1 times of amount, 2 times of amounts, 3 times of amounts) ethyl acetate extraction respectively 1 time again, measure total coumarins content in the extraction phase and calculate the retention rate of coumarin, to determine the ethyl acetate consumption, it the results are shown in Table 19.
Table 19 ethyl acetate consumption is investigated the result
| Ethyl acetate consumption (doubly) | Total coumarins content (%) in the extraction phase | The retention rate (%) of total legumin in the extraction phase |
| 1 | 39.46 | 65.52 |
| 2 | 42.39 | 68.05 |
| 3 | 43.17 | 68.48 |
| Material liquid | 6.82 | 100 |
The above results shows: along with the increase of ethyl acetate consumption, total coumarins content increases gradually in the extraction phase, but the ethyl acetate consumption is 2 times of amounts when above, and the content of coumarin and retention rate increase are no longer obvious, therefore, determine to adopt the ethyl acetate of 2 times of amounts to extract.
D ethyl acetate extraction number of times is investigated
Get totally 3 parts of above-mentioned sample liquid (2g/ml), each 20ml, add petroleum ether 40ml extraction 1 time respectively, discard extraction phase, raffinate phase adds ethyl acetate and extracts respectively 1 time, 2 times, 3 times, adds ethyl acetate 40ml at every turn, measures total coumarins content in the extraction phase of different extraction times respectively and calculates the retention rate of total coumarins, to determine the ethyl acetate extraction number of times, it is table 20 as a result.
Table 20 ethyl acetate extraction number of times is investigated the result
| Ethyl acetate extraction number of times (inferior) | Total coumarins content (%) in the extraction phase | The retention rate (%) of total legumin in the extraction phase |
| 1 | 43.39 | 68.05 |
| 2 | 52.02 | 73.96 |
| 3 | 58.78 | 82.65 |
| 4 | 59.25 | 83.12 |
| Material liquid | 6.82 | 100 |
The above results shows: when the ethyl acetate extraction number of times is increased to more than 3 times, in the extraction phase increase of the content of total coumarins and retention rate no longer obvious, consider from saving cost and the angle of time, determine that the ethyl acetate extraction number of times is 3 times.
The investigation of E extract concentration
It is an amount of to get above-mentioned sample liquid (2g/ml), diluting respectively or being concentrated to liquor strength is that every ml is equivalent to medical material 1g, 2g, 3g, respectively get 20ml, add petroleum ether 40ml extraction 1 time respectively, discard extraction phase, raffinate phase adds ethyl acetate extraction 3 times, each ethyl acetate 40ml that adds, measure ethyl acetate extraction mutually in total coumarins content and calculate the retention rate of total coumarins, determine the optium concentration of extracting solution, the results are shown in Table 21.
The investigation result of table 21 extract concentration
| Liquor strength (g/ml) | Total coumarins content (%) in the extraction phase | The retention rate (%) of total legumin in the extraction phase |
| 1 | 46.18 | 66.30 |
| 2 | 57.03 | 85.28 |
| 3 | 53.96 | 81.03 |
| Material liquid | 6.82 | 100 |
By last table result as can be known: extract concentration is every ml medicinal liquid when being equivalent to the 2g medical material, and the content and the retention rate of angelica anomala total coumarins are higher, so determine that extract concentration is 2g/ml.
In sum, the optimum condition of angelica anomala coumarin separation purifying technique is: Radix Angelicae Dahuricae ethanol extract decompression recycling ethanol, and it is that every ml is equivalent to the 2g medical material that medicinal liquid is evaporated to concentration, puts cold, add 2 times of amount petroleum ether, extract 1 time, discard petroleum ether extraction layer (being extraction phase), raffinate phase adds ethyl acetate extraction 3 times, 2 times of amounts of each adding ethyl acetate, merge extraction phase, the reclaim under reduced pressure ethyl acetate, promptly.
Adopt the angelica dahurica coumarin extract of the inventive method preparation, drug effect is clear and definite, the dna purity height, and the content height of total coumarins, steady quality, controllability is strong, can reach medicinal requirement.
The assay of embodiment 4 angelica dahurica coumarin extracts of the present invention
1. chromatographic condition
Chromatographic column: Aichrom Reliasil C
18Chromatographic column (4.6mm * 250mm, 5 μ m); Mobile phase: methanol-water (70: 30); Flow velocity 1.0mLmin
-1Detect wavelength: 300nm; Column temperature: 35 ℃.
2. the preparation of solution
The preparation of A reference substance solution
It is an amount of that precision takes by weighing the imperatorin reference substance, adds methanol and make solution that every 1ml contains 0.0521mg product solution in contrast.
The preparation of B sample test liquid
Take by weighing embodiment 1 angelica anomala coumarin effective site sample 0.101g (lot number: 061008) put in the 50ml measuring bottle, with dissolve with methanol and fixed to scale, precision is measured in 2ml to the 10ml measuring bottle, with methanol dilution and fixed to scale, shake up, filter with 0.45 μ m microporous filter membrane, get subsequent filtrate, promptly get the sample test liquid.
3. system suitability test
Draw reference substance solution 7 μ L, sample solution 10 μ L respectively, inject hplc determination, the record chromatogram by above chromatographic condition.Adopt above-mentioned chromatographic condition, the retention time of imperatorin is: 11.5min, and separating degree is greater than 1.5, and the separating effect of visible imperatorin is better.
The repeatability test
By above-mentioned content assaying method, to same batch sample (lot number: 061008) parallel laboratory test is 5 times, the content of imperatorin in the working sample, it the results are shown in Table 22.
Table 22 sample replica test result
The above results shows: relative deviation RSD is 0.52%＜2%, illustrates that sample test liquid repeatability is good when measuring.
5. the assay of total coumarins and imperatorin in the Radix Angelicae Dahuricae effective site
Get 10 batch samples (lot number: 061006,061008,061010,061012,061014,061016,061018,061020,061022,061024), by UV content assaying method and above-mentioned HPLC content assaying method the angelica anomala coumarin extract is carried out total coumarins and imperatorin assay, the results are shown in Table 23.
Table 23 Radix Angelicae Dahuricae effective part extract assay result
| Lot number | Total coumarins content (%) | Imperatorin content (mg/g) |
| 061006 | 54.955 | 85.295 |
| 061008 | 55.875 | 85.482 |
| 061010 | 56.692 | 86.369 |
| 061012 | 56.474 | 86.250 |
| 061014 | 55.837 | 85.479 |
| 061016 | 57.382 | 87.973 |
| 061018 | 56.297 | 86.213 |
| 061020 | 55.285 | 85.125 |
| 061022 | 54.567 | 84.440 |
| 061024 | 55.129 | 85.164 |
| Meansigma methods | 55.849 | 85.779 |
Above result shows: preparation process condition is more stable, each batch angelica anomala coumarin effective site changes of contents is less, total coumarins content meansigma methods is: 55.849%, the meansigma methods of imperatorin is: 85.779%, in 80% of above-mentioned meansigma methods, the content of total coumarins should be not less than 44.672mg/g in the tentative angelica anomala medical material, and the content of imperatorin should be not less than 68.623mg/g.
The HPLC finger printing of embodiment 5 angelica anomala coumarin effective sites
(1) formulations prepared from solutions
1. the preparation of reference substance solution
It is an amount of that precision takes by weighing the imperatorin reference substance, adds methanol and make solution that every 1ml contains imperatorin 0.0521mg product solution in contrast.
2. the preparation of sample test liquid
(lot number: 061008) 0.1g puts in the 50ml measuring bottle, adds dissolve with methanol and fixed to scale, with the microporous filter membrane filtration of 0.45 μ m, gets subsequent filtrate, promptly gets the sample test liquid to get angelica anomala coumarin effective site sample.
(2) foundation of chromatographic process
The optimum chromatogram condition of angelica anomala coumarin effective site HPLC finger printing research is: chromatographic column: Aichrom Reliasil C
18Chromatographic column (4.6mm * 250mm, 5 μ m), mobile phase: methanol, water and oxolane gradient elution, column temperature: 40 ℃, flow velocity: 1ml/min, wavelength: 300nm, sample size: 5 μ l.
(3) foundation and the analysis of angelica anomala coumarin effective site HPLC finger printing
1. the preparation of need testing solution
Get angelica anomala coumarin effective site sample (lot number: 061006,061008,061010,061012,061014,061016,061018,061020,061022,061024) each about 0.1g, put in the 50ml measuring bottle, add dissolve with methanol and fixed to scale, shake up, microporous filter membrane with 0.45 μ m filters, get subsequent filtrate, promptly get the sample test liquid.
2. the foundation and the similarity evaluation of angelica anomala coumarin effective site finger printing
The a object of reference is selected
Imperatorin is the Coumarins composition, also be one of main pharmacodynamics composition in the Radix Angelicae Dahuricae simultaneously, content is bigger, structure is clear and definite, in sample HPLC chromatogram, the chromatographic peak of imperatorin and adjacent chromatographic peak separating degree are good, and peak position is more placed in the middle, be the common chromatographic peak of all samples simultaneously, therefore select the leading indicator composition imperatorin (No. 9 chromatographic peaks) of coumarin to be the object of reference of Radix Angelicae Dahuricae effective site finger printing.
Determining of the total peak of b
By above-mentioned experimental technique and chromatographic condition 10 batch samples are measured, relatively the chromatogram of each sample is found out its metastable total peak, with imperatorin is with reference to peak (No. 9 peaks), other peak is demarcated in order with Arabic numerals, demarcates 14 total fingerprint peakses altogether, sees Fig. 1.
The comparison of total peak relative retention time of c and relative peak area
Carry out 10 batch sample analyses, as the reference peak, the peak area of each fingerprint peaks and relative peak area, retention time and relative retention time all have repeatability preferably with No. 9 peaks, and the RSD value all is lower than 3%.The average relative retention time (min) at total peak is: 7.911,9.731,13.390,16.59,19.997,23.964,29.512,30.271,31.949,34.525,37.298,39.161,39.957,42.096, see Fig. 1.
The foundation of d finger printing
10 batch sample HPLC chromatogram data of gained are had the collection of illustrative plates match with " 2004 editions A of chromatographic fingerprints of Chinese materia medica similarity evaluation system " that Chinese Pharmacopoeia Commission issues, the results are shown in Figure 2.
By last figure result as can be known: all have higher similarity between the peak number of the HPLC chromatographic peak of 10 batch samples, peak position, each peak area.
E finger printing similarity evaluation
The similarity evaluation that " 2004 editions A of chromatographic fingerprints of Chinese materia medica similarity evaluation system " that 10 batch sample HPLC chromatogram data of gained are issued with Chinese Pharmacopoeia Commission compose entirely the results are shown in Table 24.
The similarity result of table 24 Radix Angelicae Dahuricae effective site intermediate finger printing
| ? | S1 | S2 | S3 | S4 | S5 | S6 | S7 | S8 | S9 | S10 | Reference fingerprint |
| S1 | 1 | 0.985 | 0.996 | 0.997 | 0.988 | 0.993 | 0.994 | 0.996 | 0.997 | 0.997 | 0.998 |
| S2 | 0.985 | 1 | 0.984 | 0.986 | 0.998 | 0.983 | 0.98 | 0.99 | 0.984 | 0.987 | 0.991 |
| S3 | 0.996 | 0.984 | 1 | 0.998 | 0.984 | 0.994 | 0.999 | 0.993 | 0.995 | 0.998 | 0.998 |
| S4 | 0.997 | 0.986 | 0.998 | 1 | 0.987 | 0.994 | 0.996 | 0.995 | 0.998 | 0.998 | 0.999 |
| S5 | 0.988 | 0.998 | 0.984 | 0.987 | 1 | 0.979 | 0.98 | 0.989 | 0.985 | 0.987 | 0.991 |
| S6 | 0.993 | 0.983 | 0.994 | 0.994 | 0.979 | 1 | 0.993 | 0.992 | 0.996 | 0.993 | 0.996 |
| S7 | 0.994 | 0.98 | 0.999 | 0.996 | 0.98 | 0.993 | 1 | 0.99 | 0.994 | 0.996 | 0.996 |
| S8 | 0.996 | 0.99 | 0.993 | 0.995 | 0.989 | 0.992 | 0.99 | 1 | 0.992 | 0.997 | 0.997 |
| S9 | 0.997 | 0.984 | 0.995 | 0.998 | 0.985 | 0.996 | 0.994 | 0.992 | 1 | 0.995 | 0.997 |
| S10 | 0.997 | 0.987 | 0.998 | 0.998 | 0.987 | 0.993 | 0.996 | 0.997 | 0.995 | 1 | 0.999 |
| Reference fingerprint | 0.998 | 0.991 | 0.998 | 0.999 | 0.991 | 0.996 | 0.996 | 0.997 | 0.997 | 0.999 | 1 |
The above results shows: 10 batch sample similarity values are respectively: 0.998,0.991,0.998,0.999,0.991,0.996,0.996,0.997,0.997,0.999,1 (with reference to collection of illustrative plates), 10 batch sample correlation coefficient＞0.99 have similarity preferably.
This law is chosen 10 batch samples and is set up common pattern, it is representative and scientific to have guaranteed that preferably common pattern is set up, main peaks group's overall diagram looks basically identical in the sample finger printing, though the relative ratio of each component content has difference slightly in the different batches angelica anomala coumarin extract, but similarity result of calculation shows, 10 batch sample similarities are higher, similarity result of calculation is all greater than 0.99, difference is not obvious between illustrating batch, and this finger printing can more comprehensively be controlled the quality of angelica anomala effective site.
Prove by the foregoing description, adopt the angelica dahurica coumarin extract of the inventive method preparation, compare with the conditional parameter of other extracting method and selection, purity is higher, the content height of total coumarins, and quality is more stable, can reach medicinal requirement.
Below prove beneficial effect of the present invention by concrete pharmacodynamics test.
Test example 1 angelica anomala total coumarins is to the influence of low 5-HT migraine animal model
1, material
1.1 the medicine angelica anomala is purchased in Radix Angelicae Dahuricae GAP production base, Suining, Sichuan, angelica anomala total coumarins extractum (is prepared by Chinese medicine preparation teaching and research room of Xihua Univ, the total coumarins assay is 55.8%), it is standby to be made into (containing 0.5% tween 80 hydrotropy) suspension of desired concn with distilled water during experiment.
1.2 the reagent ergotamine and caffeine tablet, Shanghai Pharmaceutical's letter friendship pharmacy head factory, product batch number 20060401; The reserpine injection, Guangdong Bangmin Pharmaceutical Co., Ltd., lot number 060308; All the other medicine and reagents are homemade analytical pure.
1.3 instrument ACS-AA type electronic weighing scale, Shanghai Yousheng Balance Co., Ltd.; FA1004N electronic balance (ten thousand/), French Sai Duolisi company.
1.4 animal SD rat, male and female dual-purpose, body weight 180～200g; Kunming mouse, female entirely, body weight 18～20g; Animal quality certification scxk (river 2004-11) is provided by Chengdu University of Traditional Chinese Medicine's Experimental Animal Center.
2, method:
2.1 the angelica anomala total coumarins is to the influence of the low 5-HT type migraine animal model of reserpinization
(pain threshold is greater than 10s to screen 60 of qualified female Kunming mouses that meet experiment with hot plate method, less than 30s), be divided into 5 groups by the body weight stratified random, 12 every group, be respectively blank group, model group, angelica anomala total coumarins high dose group, middle dosage group, low dose group.Except that the blank group, all the other respectively organize every equal subcutaneous injection of reserpine 10mg/kg of mice, once a day, altogether behind the 14d, experimental group gives angelica anomala total coumarins suspension oral gavage (dosage sees Table 7), blank group and model group give isometric(al) distilled water (containing 0.5% tween 80), once a day, and continuous 3d.1h after the 3d administration, hot plate method is surveyed pain threshold.
2.2 statistical method carries out with " Chinese medicine encyclopedia medicostatistics " statistical package PEMS3.1.Relatively adopt t check in twos, many groups are relatively adopted variance analysis, and homogeneity of variance is with multiple comparisons methods such as LSD or Tukey, and heterogeneity of variance is with Tamhane ' s T2 multiple comparisons method.
3, result
Table 25 angelica anomala total coumarins to the influence of the low 5-HT type Nerve in Migraine Model mice pain threshold of reserpinization (x ± s, n=10)
| Group | Number of animals (only) | Dosage (g/kg) | Pain threshold (second) |
| The blank group | 12 | - | 16.17±4.47 |
| Model control group | 12 | - | 15.83±5.17 |
| The total coumarin |
12 | 0.72Qd×3 | 21.67±5.28 * |
| The total coumarin |
12 | 0.36Qd×3 | 18.83±6.63 |
| The total coumarin |
12 | 0.18Qd×3 | 17.42±7.57 |
Annotate: compare with model group,
*P＜0.05,
*P＜0.01
Table 25 result shows: angelica anomala total coumarins high dose group can obviously improve the pain threshold of model mice, and relatively there were significant differences (P＜0.05) with model group.
4, discuss
This experimentation shows: on the low 5-HT migraine mouse model of monoamine transmitters depletor reserpine induction, the angelica anomala total coumarins can obviously improve the pain threshold (P＜0.05) of mice under 0.72g/kg dosage, have clear and definite analgesic activity.
Test example 2 angelica anomala total coumarinss are to the influence of serotonin and monoamine oxidase, MAO
1, material
1.1 the medicine angelica anomala is purchased in Radix Angelicae Dahuricae GAP production base, Suining, Sichuan, angelica anomala total coumarins extractum (is prepared by Chinese medicine preparation teaching and research room of Xihua Univ, the total coumarins assay is 55.8%), it is standby to be made into (containing 0.5% tween 80 hydrotropy) suspension of desired concn with distilled water during experiment.
1.2 reagent 5-hydroxy tryptamine creatinine sulfate, the chemical reagent purchasing station import packing of China Drug Co. Shanghai; All the other reagent are import or homemade analytical pure.Monoamine oxidase, MAO, building up bio-engineering research by Nanjing is provided.
1.3 instrument
The 960CRT spectrofluorophotometer, Shanghai Precision Scientific Apparatus Co., Ltd; 751 ultraviolet spectrophotometers, Shanghai Precision Scientific Apparatus Co., Ltd; Heraeus BioFuge pico desk centrifuge (Germany); Heraeus BioFuge stratos low-temperature and high-speed centrifuge (Germany); The full-automatic ice machine of SANYO (Japan); SANYO ultra cold storage freezer (Japan); The ultrasound wave refiner, Zhejiang Province three door ultrasound wave instrument plants; ACS-AA type electronic weighing scale, Shanghai Yousheng Balance Co., Ltd.; FA1004N electronic balance (ten thousand/), French Sai Duolisi company.
1.4 the animal Kunming mouse, male and female dual-purpose, body weight 18～20g; Animal quality certification scxk (river 2004-11) is provided by Chengdu University of Traditional Chinese Medicine's Experimental Animal Center.
2, method
2.1 experimental technique
Get 60 of Kunming mouses, be divided into 5 groups, 12 every group, be respectively blank group, model group, angelica anomala total coumarins high dose group, middle dosage group, low dose group by the body weight stratified random.Except that the blank group, all the other respectively organize every equal subcutaneous injection of reserpine 10mg/kg of mice, altogether 14d.From 7d, experimental group gives angelica anomala total coumarins suspension oral gavage (dosage sees Table 14), and blank group and model group are given isometric(al) distilled water (containing 0.5% tween 80), continuously 7d.1h after the 14d administration plucks eyeball and gets blood, then takes off cervical vertebra and puts to death animal, separates cerebral tissue rapidly.
2.2 the detection method that 5-HT and monoamine oxidase MAO are measured in blood and the brain
5-HT presses literature method mensuration in blood and the brain, and monoamine oxidase, MAO is measured and undertaken by the test kit description.
The mensuration concrete grammar of 5-HT is as follows in the brain:
Put a small amount of acidify n-butyl alcohol in homogenizer.Get and put into homogenizer after tissue is weighed, in ice bath, prepare homogenate.Homogenate is transferred to graduated centrifuge tube, mends to 4ml with the acidify n-butyl alcohol.Vibration 5min.Centrifugal 5min gets supernatant 2.5ml and puts into another centrifuge tube.Every pipe adds normal heptane 5ml and 0.1MHCL 1.2ml.Vibration 5min.Centrifugal 5min, this moment, solution was divided into biphasely, got 0.5ml respectively in another test tube from aqueous phase.Add 0.5% cysteine 0.1ml, add 0.004%OPT 3ml again, behind the boiling water bath 10min, the 5-HT fluorescence intensity is measured in the cold water cooling at the 480nm/365nm place.The following formula of fluorescence intensity A value substitution with the 5-HT that records gets final product then:
Detect 5-HT content in the blood with method.
2.3 statistical method carries out with " Chinese medicine encyclopedia medicostatistics " statistical package PEMS3.1.Relatively adopt t check in twos, many groups are relatively adopted variance analysis, and homogeneity of variance is with multiple comparisons methods such as LSD or Tukey, and heterogeneity of variance is with Tamhane ' s T2 multiple comparisons method.
3, result
Table 26 angelica anomala total coumarins is to the influence of 5-HT in the Nerve in Migraine Model mouse brain (x ± s)
| Group | Number of animals (only) | Dosage (g/kg) | 5-HT content (ng/g) |
| The blank group | 12 | - | 0.0630±0.0171 ** |
| Model control group | 12 | - | 0.0374±0.0153 |
| The total coumarin |
12 | 0.72Qd×7 | 0.0531±0.0157 * |
| The total coumarin |
12 | 0.36Qd×7 | 0.0513±0.0010 * |
| The total coumarin |
12 | 0.18Qd×7 | 0.0419±0.0200 |
Annotate: compare * P＜0.05 with model group
Table 26 result shows: but angelica anomala total coumarins 5-HT content in the elevation model animal brain under 0.72g/kg and 0.36g/kg dosage relatively has notable difference (P＜0.05) with model group.
Table 27 angelica anomala total coumarins is to the influence of 5-HT in the Nerve in Migraine Model mice blood (x ± s)
| Group | Number of animals (only) | Dosage (g/kg) | 5-HT content (ng/g) |
| The blank group | 12 | - | 0.1358±0.0371 ** |
| Model control group | 12 | - | 0.0812±0.0166 |
| The total coumarin |
12 | 0.72Qd×7 | 0.1209±0.0208 ** |
| The total coumarin |
12 | 0.36Qd×7 | 0.1149±0.0415 * |
| The total coumarin |
12 | 0.18Qd×7 | 0.1014±0.0200 |
Annotate: compare * * P＜0.01, * P＜0.05 with model group
Table 27 result shows: but angelica anomala total coumarins 5-HT content in the elevation model animal blood under 0.72g/kg and 0.36g/kg dosage relatively has notable difference (P＜0.05, P＜0.01) with model group.
Table 28 angelica anomala total coumarins is to the influence of monoamine oxidase, MAO in the Nerve in Migraine Model mouse brain (x ± s)
| Group | Number of animals (only) | Dosage (g/kg) | MAO content (U/mgprot) |
| The blank group | 10 | - | 17.49±9.39 |
| Model control group | 10 | - | 19.34±6.75 |
| The total coumarin |
10 | 0.72Qd×7 | 15.09±4.65 |
| The total coumarin |
10 | 0.36Qd×7 | 18.25±8.18 |
Annotate: compare P＞0.05 with model group
Table 28 result shows: the angelica anomala total coumarins under 0.72g/kg and 0.36g/kg dosage to the animal pattern brain in the MAO activity all do not have obvious influence (P＞0.05).
Table 29 angelica anomala total coumarins is to the influence of monoamine oxidase, MAO in the Nerve in Migraine Model mice blood (x ± s)
| Group | Number of animals (only) | Dosage (g/kg) | MAO content (U/ml) |
| The blank group | 10 | - | 7.97±2.79 |
| Model control group | 10 | - | 8.84±2.12 |
| The total coumarin |
10 | 0.72Qd×7 | 10.04±2.79 |
| The total coumarin |
10 | 0.36Qd×7 | 9.13±2.28 |
Annotate: compare P＞0.05 with model group
Table 29 result shows: the angelica anomala total coumarins under 0.72g/kg and 0.36g/kg dosage to animal pattern blood in the MAO activity all do not have obvious influence (P＞0.05).
4, discuss
In migrainous onset theory, (5-hydroxvtryptamine, exhaustion hypothesis 5-HT) is occupied important position to biochemical source 5-hydroxy tryptamine., make platelet discharge 5-HT at first because of but specific effect in some reason migraineur blood raises in hematoblastic 5-HT releasing factor.The 5-HT that discharges shrinks intracranial vessel, causes the prodrome of ischemia.Intracranial vessel shrinks and causes stress simultaneously, and exciting sympathetic nerves makes epinephrine and free fatty increase in the blood, acts on platelet, produces thromboxane A2 (Thromboxan A
2, TXA
2), TXA
2Mainly synthetic in platelet microsome, have very strong vasoconstrictive effect, further promote the effect that 5-HT shrinks intracranial vessel.(its result exhausts 5-HT to the 5-HT that raises, and causes to Secondary cases the overdistension of blood vessel, thereby produces pulsatile headache for monoamine oxidase a, MAO-A) constantly decomposition of institute by monoamine oxidase, MAO A.The foreign scholar finds in migraine phase patient's blood plasma that 5-HT content reduces, and in the migraine clinical treatment the sure curative effect of two class 5-HT receptor stimulating agents also part proved the reasonability of this theory.Therefore, investigated of the influence of angelica anomala total coumarins to 5-HT and monoamine oxidase, MAO.
Adopt the low experimental migraine mouse model of 5-HT of reserpinization to experimentize, the result shows: the angelica anomala total coumarins obviously reaches 5-HT level (P＜0.05, P＜0.01) in the blood in the elevation model mouse brain under 0.72g/kg and 0.36g/kg dosage; But in the research of monoamine oxidase, MAO, each dosage group of angelica anomala total coumarins does not have obvious influence to activity of monoamine oxidase in animal pattern midbrain and the blood, and the effect of 5-HT in angelica anomala total coumarins elevation model animal brain and the blood is described.
Above-mentioned test of pesticide effectiveness explanation, adopt the highly purified total coumarins of extracting method preparation of the present invention, steady quality, has the 5-hydroxy tryptamine regulating action, except the treatment migraine, also can treat 5-hydroxy tryptamine and reduce other disease that causes, drug effect is clear and definite, indication is with strong points, provides a kind of new selection for clinical.
Claims (5)
1. angelica dahurica coumarin extract, it is characterized in that: the content of total coumarins should be not less than 44.672mg/g, and the content of imperatorin should be not less than 68.623mg/g; Wherein, the preparation method of angelica dahurica coumarin extract is:
A, get angelica root, granularity is 20 orders, adds 75% alcohol heat reflux and extracts, and the extracting solution decompression recycling ethanol concentrates;
B, a step is concentrated adds petroleum ether extraction, discards the petroleum ether extraction layer; Raffinate phase adds ethyl acetate extraction, merges extraction phase, and the reclaim under reduced pressure ethyl acetate promptly gets angelica dahurica coumarin extract.
2. angelica dahurica coumarin extract according to claim 1 is characterized in that: its HPLC chromatograph contains 14 total fingerprint peakses as shown in Figure 1;
Its chromatographic condition is:
Chromatographic column: Aichrom Reliasil C
18Chromatographic column 4.6mm * 250mm, 5 μ m, mobile phase: methanol, water and oxolane gradient elution, column temperature: 40 ℃, flow velocity: 1ml/min, wavelength: 300nm, sample size: 5 μ l, methanol, water and oxolane gradient elution time and ratio are:
Elution time: 0min, the percentage by weight of water, oxolane, methanol is respectively: water accounts for 60%, oxolane 8%, methanol 32%; Elution time: 10min, the percentage by weight of water, oxolane, methanol is respectively: water accounts for 60%, oxolane 8%, methanol 32%; Elution time 50min, the percentage by weight of water, oxolane, methanol is respectively: water accounts for 20%, oxolane 16%, methanol 64%.
3. angelica dahurica coumarin extract according to claim 2 is characterized in that: the average relative retention time min at described total peak is: 7.911,9.731,13.390,16.59,19.997,23.964,29.512,30.271,31.949,34.525,37.298,39.161,39.957,42.096.
4. the quality determining method of the described angelica dahurica coumarin extract of claim 1, it is to adopt the HPLC finger printing to carry out quality control, wherein, chromatographic condition is:
Chromatographic column: Aichrom Reliasil C
18Chromatographic column 4.6mm * 250mm, 5 μ m, mobile phase: methanol, water and oxolane gradient elution, column temperature: 40 ℃, flow velocity: 1ml/min, wavelength: 300nm, sample size: 5 μ l, methanol, water and oxolane gradient elution time and ratio are:
Elution time: 0min, the percentage by weight of water, oxolane, methanol is respectively: water accounts for 60%, oxolane 8%, methanol 32%; Elution time: 10min, the percentage by weight of water, oxolane, methanol is respectively: water accounts for 60%, oxolane 8%, methanol 32%; Elution time 50min, the percentage by weight of water, oxolane, methanol is respectively: water accounts for 20%, oxolane 16%, methanol 64%.
5. a method for preparing each described angelica dahurica coumarin extract of claim 1-3 comprises the steps:
A, get angelica root, granularity is 20 orders, adds 75% alcohol heat reflux and extracts, and the extracting solution decompression recycling ethanol concentrates;
B, a step is concentrated adds petroleum ether extraction, discards the petroleum ether extraction layer; Raffinate phase adds ethyl acetate extraction, merges extraction phase, the reclaim under reduced pressure ethyl acetate, promptly.
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| CN103356722A (en) * | 2013-07-31 | 2013-10-23 | 西华大学 | Angelica dahurica total coumarin solid dispersion and preparation method therefor |
| CN105699564A (en) * | 2014-11-28 | 2016-06-22 | 舒泰神（北京）生物制药股份有限公司 | Construction method of fingerprint of sweet clover medicinal material and standard fingerprint thereof |
| CN107880007A (en) * | 2017-08-28 | 2018-04-06 | 成都医学院 | A kind of method of the extraction separation and purification Scopoletin from erycibe schmidtii Craib |
| CN108152392B (en) * | 2017-12-08 | 2020-09-08 | 宋瑞 | Detection method of angelica dahurica coumarin in veterinary medicine |
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| CN110824029A (en) * | 2018-08-13 | 2020-02-21 | 成都中医药大学 | UPLC and one-test-multiple-evaluation method based detection of content of coumarins in radix angelicae medicinal material |
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