CN1904591A - Determining method of artemislnin content in artemisia apiacea - Google Patents
Determining method of artemislnin content in artemisia apiacea Download PDFInfo
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- CN1904591A CN1904591A CNA2006100950175A CN200610095017A CN1904591A CN 1904591 A CN1904591 A CN 1904591A CN A2006100950175 A CNA2006100950175 A CN A2006100950175A CN 200610095017 A CN200610095017 A CN 200610095017A CN 1904591 A CN1904591 A CN 1904591A
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Abstract
The invention relates to a test method for arteannuin content in southernwood that needs not fully distill the arteannuin in southernwood. It adopts ultraviolet spectrum luminosity analysis method to test the arteannuin content data M (mg/g), and substituting to the linear model built up from the corresponding relationship of arteannuin content data tested by liquid chromatography after fully distilling the arteannuin, and calculating the actual content X (mg/g) in southernwood. The invention simplifies the operation process, shortens distilling time and could finish test in 1.5h. The actual content data of arteannuin is reliable. The invention could also realize rapid test content of arteannuin in southernwood purchasing locale.
Description
Technical field
The present invention relates to the assay method of artemislnin content in a kind of sweet wormwood, particularly a kind of assay method that adopts artemislnin content in the ultraviolet spectro-photometric analysis sweet wormwood.
Background technology
Qinghaosu and with the qinghaosu be the synthetic artemisinin-based antimalarial drugs such as Artemether, dihydroartemisinine and Artesunate of raw material by world health organisation recommendations for replacing the safe and effective medicine of quinine treatment malaria at present.Because the amount widely different (2 ‰～12 ‰) of the contained qinghaosu of sweet wormwood medicinal material that different regions produce, the significant ecological region of tool, therefore must carry out quality control to the sweet wormwood medicinal material, particularly in the medicinal material purchase, supply, need both sides all to need method the quality of sweet wormwood is estimated fast, promptly be badly in need of the method for fast measuring artemislnin content.
Domestic and international existing artemislnin content assay method has multiple at present.As high performance liquid chromatography, thin-layered chromatography, supercritical fluid chromatography, luminosity enzyme-linked immunosorbent assay, these methods are mainly used in the mensuration of the qinghaosu that content is higher, purity is higher, but require instrument and equipment height, complicated operation, length consuming time, be not suitable for on-the-spot quality assessment fast; Also having ultraviolet spectrophotometry in addition, mainly is to detect at the very high qinghaosu of purity, though easy and simple to handle, length consuming time also is not suitable for being directly used in on-the-spot quality assessment fast.
Summary of the invention
The object of the present invention is to provide the method for sweet wormwood content in a kind of easy and simple to handle, fast detecting sweet wormwood.
The object of the present invention is achieved like this: the assay method of artemislnin content in a kind of sweet wormwood, it is characterized in that: not exclusively extract the qinghaosu in the sweet wormwood to be measured, adopt the ultraviolet spectro-photometric analysis method to measure artemislnin content data M (mg/g), substitution by with qinghaosu is extracted fully the linear model that the corresponding relation that adopts the artemislnin content data that liquid phase chromatography measures in the back is set up, calculate the actual content X (mg/g) of the qinghaosu in the sweet wormwood of surveying.
Above-mentioned qinghaosu not exclusively extracts and adopts the ultrasonic extraction method to extract; Above-mentioned qinghaosu extracts fully and adopts conventional soxhlet extraction to extract.
The linear model that the corresponding relation of above-mentioned artemislnin content data is set up is X=(M+0.3995)/0.748.
The incomplete extraction step of above-mentioned qinghaosu is as follows: weighing 0.5 ± 0.01g, and 60～80 orders sweet wormwood powder to be measured sample adds the 15ml sherwood oil therein, is under 30～60 ℃ of conditions in temperature, ultrasonic 12～17min, record sherwood oil volume V; Precision is measured the petroleum ether solution 0.3ml that crosses through sonicated in 10ml scale test tube, puts into boiling water and volatilizees fully until sherwood oil; The residue of retaining is derived: 95% ethanol that adds 3ml dissolves again, and uses 0.2%NaOH solution constant volume to 10ml, mixing, and water-bath 30min under 50 ± 1 ℃ of temperature gets testing sample solution.
It is being reference through the blank solution of derivation process that above-mentioned ultraviolet spectro-photometric analysis method measures artemislnin content data M mg/g, at UV spectrophotometer measuring wavelength 292nm place, measure qinghaosu standard solution and testing sample solution absorbance, record absorbance and be respectively A through derivation process
StandardAnd A
Sample
Calculate artemislnin content with criterion keying method, computing formula is:
M=(A
Sample* V * 2)/(A
Standard* 3)
Wherein, A
SampleAbsorbance for the testing sample that records; V is the volume of ultrasonic back sample sherwood oil, in ml; A
StandardAbsorbance for 0.1mg/ml qinghaosu standard items.
The derivation process of above-mentioned blank solution:, use 0.2%NaOH solution constant volume to 10ml, mixing, water-bath 30min under 50 ± 1 ℃ of temperature again with the 3ml95% ethanolic solution; Deriving of described qinghaosu standard solution: will contain and add the 2ml95% ethanolic solution in the 1ml solution of qinghaosu standard items 0.1mg/mL, with 0.2%NaOH solution constant volume to 10ml, mixing, water-bath 30min under 50 ± 1 ℃ of temperature again, be cooled to room temperature then, get with 0.22 μ m membrane filtration.
The present invention is in the ultrasonic mode that replaces traditional long-time backflow (needing 15～30 hours usually) to extract sweet wormwood of not exclusively extracting fast, simplified running program, shortened extraction time, and directly measure qinghaosu in the sweet wormwood crude extract with the ultraviolet-visible spectrophotometric method, in 1.5 hours, just can finish the assay of qinghaosu, required instrument is simple, easy to operate, saved reagent, reduced pollution: the dependent linearity model that detects data owing to Soxhlet (fully) extraction-high performance liquid chromatography (HPLC) of the ultraviolet fast detecting data of setting up qinghaosu and qinghaosu, guaranteed the reliability of the artemislnin content data that computing obtains, therefore, the present invention can really be implemented in the on-the-spot fast detecting of carrying out artemislnin content of sweet wormwood medicinal material purchase.
Description of drawings
Fig. 1: the present invention not exclusively extracts-and ultraviolet spectro-photometric analysis method and Soxhlet (fully) extraction-high performance liquid chromatography detect the linear regression graph of the artemislnin content data of surveying.
Fig. 2: the incomplete extraction time of extracting and the change curve of artemislnin content.
Embodiment
Implementation column 1: the sweet wormwood medicinal material of choosing 22 kinds of different artemislnin contents is divided in duplicate, a employing the present invention not exclusively extracts, the ultraviolet spectro-photometric analysis method measures the artemislnin content data M, another part adopts Soxhlet Liquid Detection method to measure artemislnin content data M s, with the regretional analysis of two groups of data inlet wires, obtain its dependent linearity model then.
1, the not exclusively enforcement of extraction, ultraviolet spectro-photometric analysis method
1.1 instrument and reagent
Balance (0.01g), KQ2200B type ultrasonic cleaner, tool plug 150ml Erlenmeyer flask, tool plug 10ml scale test tube, the 25ml graduated cylinder, electric furnace, constant water bath box, 752 ultraviolet spectrophotometers, 0.22 μ m water-based filtrator, sherwood oil (30～60 ℃), 95% ethanol, 0.2% NaOH (NaOH) solution.
1.2 test procedure
1.2.1 the preparation of solution:
A, qinghaosu standard solution: precision weighing qinghaosu standard items 10mg adds the dilution of 95% dissolve with ethanol in the 10ml volumetric flask, constant volume obtains the reference substance stock solution of 1mg/ml to 10ml, and 4 ℃ of refrigerators are preserved;
B, 0.2%NaOH solution: precision weighing NaOH1g adds the 500ml dissolved in distilled water, mixing, room temperature preservation in the 500ml reagent bottle;
1.2.2 preparation blank, standard solution:
The preparation of a, blank solution: get the 3ml95% ethanolic solution and use 0.2%NaOH solution constant volume to 10ml in 10ml scale test tube, mixing promptly gets blank solution;
The preparation of b, standard solution: draw stock solution 1ml in 10ml scale test tube, mixing promptly gets the 0.1mg/ml standard solution.
1.2.3 the preparation of sweet wormwood sample solution.
A, get sweet wormwood and put into comminutor and pulverize, cross 80 and 60 mesh sieves respectively, obtain 60～80 order sweet wormwood powder;
B, weighing 0.5 ± 0.01g sweet wormwood sample (60～80 order) add 15ml sherwood oil (30～60 ℃), ultrasonic 15min in 150ml tool plug awl;
C, pour petroleum ether solution into graduated cylinder after ultrasonic, record sherwood oil volume V;
D, precision are measured the petroleum ether solution 0.3ml that crosses through sonicated in 10ml scale test tube, and the boiling water of putting into no longer heating volatilizes.
1.2.4 qinghaosu is derived
A, in the test tube that 1.2.3, d volatilize, add 95% ethanol of 3ml, dissolved residue, 0.2%NaOH solution constant volume be to 10ml, mixing, 50 ± 1 ℃ of water-bath 30min;
B, blank solution are derived: get blank solution, 50 ± 1 ℃ of water-bath 30min;
C, standard solution are derived: get 0.1mg/ml standard solution 1ml in the 10ml test tube, add the 2ml95% ethanolic solution, 0.2%NaOH solution constant volume is to 10ml, mixing, 50 ± 1 ℃ of water-bath 30min.
Cold water cool to room temperature after deriving, 0.22 μ m membrane filtration, filtrate is used for ultraviolet spectrophotometer and measures absorbance.
1.2.5 qinghaosu detects
With the blank solution is reference, detecting wavelength 292nm place, measures qinghaosu standard solution and sweet wormwood sample solution absorbance, records absorbance and is respectively A
StandardAnd A
Sample
1.3 artemislnin content calculates
Calculate artemislnin content with criterion keying method, computing formula is:
M (mg/g)=(A
Sample* V * 2)/(A
Standard* 3)
In the above-mentioned formula: A
SampleBe sweet wormwood sample solution measured value; V is the volume of ultrasonic back sweet wormwood sample sherwood oil, in ml, and A
StandardBe 0.1mg/ml qinghaosu standard solution absorbance.
Artemislnin content M data by 22 kinds of measured different sweet wormwood medicinal materials of above-mentioned detection method see Table 1.
The measurement result of table 1 sample
| Sample number into spectrum | Soxhlet liquid content Ms (mg/g) | Ultrasonic ultraviolet content M (mg/g) |
| 1 | 9.29 | 6.77 |
| 2 | 7.83 | 5.94 |
| 3 | 7.8 | 6.01 |
| 4 | 7.2 | 4.7 |
| 5 | 10.17 | 7.52 |
| 6 | 4.77 | 3.27 |
| 7 | 6.62 | 4.53 |
| 8 | 10.77 | 7.81 |
| 9 | 7.61 | 4.65 |
| 10 | 6.54 | 4.6 |
| 11 | 8.19 | 5.29 |
| 12 | 6.04 | 4.27 |
| 13 | 5.5 | 3.68 |
| 14 | 5.37 | 3.89 |
| 15 | 7.22 | 4.71 |
| 16 | 7.64 | 4.81 |
| 17 | 7.5 | 4.75 |
| 18 | 5.54 | 3.75 |
| 19 | 7.38 | 5.37 |
| 20 | 5.14 | 3.65 |
| 21 | 7.49 | 5.07 |
| 22 | 8.15 | 5.54 |
2, the enforcement of Soxhlet Liquid Detection method
2.1 instrument and reagent
Apparatus,Soxhlet's, U.S. Agilent 1100 high performance liquid chromatographs, anhydrous sodium acetate, glacial acetic acid, acetonitrile, distilled water.
2.2 test procedure
2.2.1 the sweet wormwood Soxhlet is extracted: get 1g sweet wormwood sample, wrap with filter paper, in apparatus,Soxhlet's, add sherwood oil (30～60 ℃) 70ml (can add a certain amount of sherwood oil as one sees fit in the water-bath process), 65 ± 1 ℃ of cable-styled extractions of water-bath 30 hours, extract finish after, the volume V of sample solution, get the 0.5ml sample solution, in the boiling water that does not heat, volatilize.
2.2.2 deriving of qinghaosu: in the test tube that volatilizes, add 95% ethanol of 3ml, dissolved residue, 0.2%NaOH constant volume be to the 10ml mixing, 50 ± 1 ℃ of water-bath 30min.
2.2.3 the detection of qinghaosu: the back cold water cool to room temperature of deriving, get quantitative derivative solution and mix with 0.08mol/l acetum equal-volume, sample introduction 50 μ l, HPLC detects.
Chromatographic condition: chromatographic column: Agilent Eclipse XDB C8 post (5 μ m, the acetonitrile of 4.6 * 150mm) moving phases: acetate buffer solution (adjust pH is 5.8)=23: 77 temperature: 30 ℃ of flow velocitys: 1ml/min wavelength: 260nm
2.3 the processing of testing result:
The artemislnin content computing formula:
Ms (mg/g)=(A
Sample* V * 2)/(A
Standard* 5)
Wherein, A
SampleBe sweet wormwood sample peak area value, V is the volume of sweet wormwood sample, A
StandardBe 0.1mg/ml qinghaosu standard items peak area value.
Artemislnin content Ms data by 22 kinds of measured different sweet wormwood medicinal materials of above-mentioned detection method see the above table 1.
The foundation of 3, not exclusively extract, ultraviolet spectro-photometric analysis method and Soxhlet Liquid Detection method being surveyed the linear model of artemislnin content data.
Two groups of data inlet wires regretional analysis with in the table 1 obtains its dependent linearity model, and promptly the real content computing formula of qinghaosu is X=(M+0.3995)/0.748 in the sweet wormwood.
In the formula: X is the real content of qinghaosu in the sweet wormwood, and unit is mg/g; M is the content of the incomplete qinghaosu that extracts, and unit is mg/g.
Embodiment 2: in the present embodiment, except that be respectively 5,12,15,20,25 the time of not exclusively extracting with a kind of sweet wormwood medicinal material, the 30min, other with embodiment 1 in not exclusively extract, the detection method of ultraviolet spectro-photometric analysis method is identical, measured same a kind of sweet wormwood medicinal material sees Table 2 in the artemislnin content data of difference extraction time condition, and not exclusively the extraction time of extracting and the change curve of artemislnin content are seen Fig. 2.As can be known from Fig. 2, in the extraction time of 12～17min scope, measured artemislnin content data are stable basically.
Table 2
| | 5 | 12 | 15 | 20 | 25 | 30 |
| Artemislnin content mg/g | 0.94 | 1.7 | 1.74 | 1.75 | 1.86 | 2.0 |
Claims (6)
1, the assay method of artemislnin content in a kind of sweet wormwood, it is characterized in that: not exclusively extract the qinghaosu in the sweet wormwood to be measured, adopt the ultraviolet spectro-photometric analysis method to measure artemislnin content data M mg/g, substitution by with qinghaosu is extracted fully the linear model that the corresponding relation that adopts the artemislnin content data that liquid phase chromatography measures in the back is set up, calculate the actual content Xmg/g of the qinghaosu in the sweet wormwood of surveying.
2, the assay method of artemislnin content in the sweet wormwood as claimed in claim 1 is characterized in that: described qinghaosu not exclusively extracts and adopts the ultrasonic extraction method to extract; Described qinghaosu extracts fully and adopts soxhlet extraction to extract.
3, the assay method of artemislnin content in the sweet wormwood as claimed in claim 2 is characterized in that: the linear model that the corresponding relation of described artemislnin content data is set up is X=(M+0.3995)/0.748.
4, the assay method of artemislnin content in the sweet wormwood as claimed in claim 1 or 2, it is characterized in that: the incomplete extraction step of described qinghaosu is as follows: weighing 0.5 ± 0.01g, 60～80 orders sweet wormwood powder to be measured sample, add the 15ml sherwood oil therein, in temperature is under 30～60 ℃ of conditions, ultrasonic 15min, record sherwood oil volume V; Precision is measured the petroleum ether solution 0.3ml that crosses through sonicated in 10ml scale test tube, puts into boiling water and volatilizees fully until sherwood oil; The residue of retaining is derived: 95% ethanol that adds 3ml dissolves again, and uses 0.2%NaOH solution constant volume to 10ml, mixing, and water-bath 30min under 50 ± 1 ℃ of temperature gets testing sample solution.
5, the assay method of artemislnin content in the sweet wormwood as claimed in claim 4, it is characterized in that: it is being reference through the blank solution of derivation process that described ultraviolet spectro-photometric analysis method measures artemislnin content data M mg/g, at UV spectrophotometer measuring wavelength 292nm place, measure qinghaosu standard solution and testing sample solution absorbance, record absorbance and be respectively A through derivation process
StandardAnd A
Sample
Calculate artemislnin content with criterion keying method, computing formula is:
M=(A
Sample* V * 2)/(A
Standard* 3)
Wherein, A
SampleAbsorbance for the testing sample that records; V is the volume of ultrasonic back sample sherwood oil, in ml; A
StandardAbsorbance for 0.1mg/ml qinghaosu standard items.
6, the assay method of artemislnin content in the sweet wormwood as claimed in claim 5, it is characterized in that: the derivation process of described blank solution: with the 3ml95% ethanolic solution, with 0.2%NaOH solution constant volume to 10ml, mixing, water-bath 30min under 50 ± 1 ℃ of temperature again; Deriving of described qinghaosu standard solution: will contain and add the 2ml95% ethanolic solution in the 1ml solution of qinghaosu standard items 0.1mg/ml, with 0.2%NaOH solution constant volume to 10ml, mixing, water-bath 30min under 50 ± 1 ℃ of temperature again, be cooled to room temperature then, get with 0.22 μ m membrane filtration.
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| CN102776212A (en) * | 2011-12-20 | 2012-11-14 | 上海交通大学 | Production method of high-artemisinin-content transgene sweet wormwood plants |
| CN102890087A (en) * | 2012-11-13 | 2013-01-23 | 宁夏医科大学 | Method for measuring artemisinin content in traditional Chinese medicinal materials such as Artemisia annua and samples containing artemisinin components |
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| CN103353498A (en) * | 2013-06-17 | 2013-10-16 | 张家港威胜生物医药有限公司 | High performance liquid chromatography method for determining content of artemisinin in artemisinin extract |
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| CN112129721A (en) * | 2020-10-23 | 2020-12-25 | 湖南星辰生物科技股份有限公司 | Ultraviolet visible light rapid detection method for artemisinin |
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Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2706166B1 (en) * | 1993-06-08 | 1995-09-15 | Archimex Ass | Artemisinin extraction process. |
| WO2003048766A2 (en) * | 2001-11-29 | 2003-06-12 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services, Centers For Disease Control And Prevention | Colorimetric artemisinin and artemisinin derivatives assay and assay kit |
| AU2002253443A1 (en) * | 2002-03-25 | 2003-10-08 | Council Of Scientific And Industrial Research | A process for isolating artemisinin from artemisia annua |
| CN1680388A (en) * | 2005-01-25 | 2005-10-12 | 商丘市明清制药有限责任公司 | Extraction of artemisine |
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2006
- 2006-08-09 CN CNB2006100950175A patent/CN100445726C/en not_active Expired - Fee Related
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| CN102776212A (en) * | 2011-12-20 | 2012-11-14 | 上海交通大学 | Production method of high-artemisinin-content transgene sweet wormwood plants |
| CN102890087A (en) * | 2012-11-13 | 2013-01-23 | 宁夏医科大学 | Method for measuring artemisinin content in traditional Chinese medicinal materials such as Artemisia annua and samples containing artemisinin components |
| CN103353498A (en) * | 2013-06-17 | 2013-10-16 | 张家港威胜生物医药有限公司 | High performance liquid chromatography method for determining content of artemisinin in artemisinin extract |
| CN103308626A (en) * | 2013-07-01 | 2013-09-18 | 浙江华立南湖制药有限公司 | Method for measuring content of impurity A in dihydroartemisinin raw material or preparation by means of HPLC (High Performance Liquid Chromatography) |
| CN104383370A (en) * | 2014-09-30 | 2015-03-04 | 成都麦的申医药科技有限公司 | Jianqu and its preparation method and quality detection method |
| CN104383370B (en) * | 2014-09-30 | 2017-12-22 | 成都麦的申医药科技有限公司 | One kind builds song and preparation method thereof and quality determining method |
| CN104237151A (en) * | 2014-10-14 | 2014-12-24 | 宁波枫康生物科技有限公司 | Mass detection method for dendrobium officinale medicine |
| CN106908433A (en) * | 2017-02-17 | 2017-06-30 | 武汉工程大学 | A kind of method that laser micro-raman spectrometry measures qinghaosu purity |
| CN106908433B (en) * | 2017-02-17 | 2019-08-16 | 武汉工程大学 | A kind of method of laser micro-raman spectrometry measurement qinghaosu purity |
| CN114200024A (en) * | 2020-09-17 | 2022-03-18 | 昆药集团股份有限公司 | Method for determining dissolution and detection of dihydroartemisinin tablet |
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