CN102053121A - Technology for rapidly detecting artemisinin content in artemisia annua - Google Patents
Technology for rapidly detecting artemisinin content in artemisia annua Download PDFInfo
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- CN102053121A CN102053121A CN2009101912583A CN200910191258A CN102053121A CN 102053121 A CN102053121 A CN 102053121A CN 2009101912583 A CN2009101912583 A CN 2009101912583A CN 200910191258 A CN200910191258 A CN 200910191258A CN 102053121 A CN102053121 A CN 102053121A
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Abstract
The invention provides technology for rapidly detecting artemisinin content. In the technology, dried leaves of sweet wormwood are taken as raw materials, and the technology comprises the steps of: rapidly extracting the artemisinin through a Soxhlet extraction method; determining chromatographic conditions of high performance liquid chromatography technology; and calculating a ratio of the artemisinin content extracted within 1 to 2 hours to the fully extracted artemisinin content so as to determine the technology for rapidly detecting the artemisinin content. The extracting conditions are that: the extraction is carried out in a water bath at the temperature of between 65 and 85 DEG C through 70 percent ethanol and the extracting time is 1 to 2 hours; after being decolored by activated carbon and deproteinized by trichloroacetic acid, the extracting solution is precipitated by 70 percent ethanol; and the supernatant is taken for high performance liquid chromatography (HPLC) detection, wherein the chromatographic conditions are that: a chromatographic column is an Agilent Eclipse XDB C8 column (5mu m, 4.6*150mm); a mobile phase is one of the following formulas: the ratio of acetonitrile to buffer solution of acetic acid is 23:77, the ratio of the acetonitrile to the buffer solution of acetic acid is 30:70 and the ratio of the acetonitrile to the buffer solution of acetic acid is 25:75; the temperature is between 25 and 35 DEG C; the flow velocity is 1 to 2 mL per minute; and the wavelength is 260nm.
Description
Technical field
The present invention relates to the qinghaosu Fast Detection Technique.Specifically relate to by soxhlet extraction and from artemisia annua, extract qinghaosu fast, and artemislnin content is carried out the technology of fast detecting with high-efficient liquid phase chromatogram technology (HPLC).Relate to the chemical detection technique field.
Background technology
Qinghaosu is a kind of brand-new antimalarial effective monomer that utilizes the distinctive plant resources artemisia annua of China to succeed in developing.Because its structure uniqueness, antimalarial mechanism is special, and the malignant malaria and the brain malaria of antagonism chloroquine have special efficacy, and characteristics of high efficiency and low toxicity is arranged, and is called " in the world unique effective malaria treatment medicine " at present by WHO.Qinghaosu is a kind of white needle-like crystals, and molecular formula is C
15H
22O
5Proved by chemical reaction, spectroscopic data and X ray single crystal diffraction method that it was a kind of sesquiterpene lactone that contains peroxy in 1976.Because it has special peroxy-radical, it is to thermally labile, is subject to wet, the influence of heat and reducing substances and decomposing.
Qinghaosu is kind of the sesquiterpene lactone with peroxide bridge and lactonic ring, does not grip structure altogether owing to it does not have, and the chemical property torpescence, and specified rate is measured and brought certain difficulty.But since qinghaosu is found,, developed and a series of analysis determining methods based on the every physicochemical property of qinghaosu through the unremitting effort of researcher.But detection method will interrelate with certain separating and extracting process, could be applied in effectively in research, the production work.It is imperative to detect transition by traditional thin-layered chromatography, ultraviolet spectrophotometry and pre-column derivatization-HPLC detection method to direct HPLC method.
Sweet wormwood have a plurality of asymmetric carbon atoms, makes synthesis step very complicated, and productive rate is low, cost is high, is difficult to be applied to suitability for industrialized production.So present medicinal qinghaosu mainly extracts from chrysanthemum punt-pole plant.Therefore, accurately the quantitative measurement artemislnin content all has important meaning for chrysanthemum punt-pole breed breeding, qinghaosu drug manufacture and pharmacological effect research.Different cultivation conditions and geographical environment artemislnin content difference are very big, when peasant household's sweet wormwood is purchased by qinghaosu manufacturing enterprise, need to measure artemislnin content, could determine purchasing price.It is longer to extract qinghaosu general extraction time in the sweet wormwood cured leaf, needs more than the 24h, and detection method is also immature.Because takeover time is more concentrated, peasant household sends sweet wormwood here and need wait 2-3 week just can obtain testing result, very inconvenience has limited the development of industry on producing, and the artemislnin content Fast Detection Technique of the present invention's research also has important value and application value in the qinghaosu industrialization development.
Summary of the invention
The objective of the invention is to set up qinghaosu high performance liquid chromatography (HPLC) Fast Detection Technique.
Content of the present invention comprises with the sweet wormwood being raw material, by soxhlet extraction rapid extraction qinghaosu, determines the chromatographic condition of high-efficient liquid phase chromatogram technology, sets up qinghaosu Fast Detection Technique system.
The present invention sets up qinghaosu Fast Detection Technique system, and detection time short (1h), reliable results is to be suitable for the mature technology that artemislnin content in the raw material detects in qinghaosu manufacturing enterprise.
(1) the Soxhlet extraction conditions of extraction qinghaosu is from sweet wormwood: 70% ethanol is in 65 ℃ of-85 ℃ of water-baths, and it is 1-2h that Soxhlet is extracted extraction time; 6-8h; 12-14h; 18-20h; 24-26h.
(2) HPLC detection chromatographic condition is: chromatographic column: Agilent Eclipse XDB C8 post (5 μ m, 4.6 * 150mm); Moving phase is following prescription choosing base one: second eyeball: acetate buffer solution=20: 80; Second eyeball: acetate buffer solution=25: 75; Second eyeball: acetate buffer solution=30: 70; Temperature: 25-35 ℃; Flow velocity: 1-2mL/min; Wavelength: 220nm.
The scheme of technical solution problem of the present invention divides following four steps to finish:
(1) the qinghaosu Soxhlet is extracted in the sweet wormwood: take by weighing the bright sample of 100g sweet wormwood, grind, sample is packed in the filter paper sleeve, sleeve is inserted in the apparatus,Soxhlet's carefully, get 300mL70% ethanol and add in the 600mL flat bottom flask, add several zeolites.1-24h is extracted in 65 ℃-85 ℃ of water-bath heating continuously, respectively at 0.5-1h, and 1-2h, 3-4h, 6-8h, 12-14h, 18-20h, 22-24h collects extract, and neutralizes with hydrochloric acid.
(2) sample is purified: extract is heated to 70 ℃ after 2% (w/v) activated carbon decolorizing, stir 30min, leave standstill the back to collect supernatant behind the centrifugal 10min of 3000r/min, 10% trichloroacetic acid with 1/5 volume removes albumen again, with the centrifugal removal metaprotein of 3000r/min, supernatant vacuum evaporation concentrates.Residue after concentrating is got the supernatant vacuum after 3000r/min is centrifugal and is concentrated into 30mL with the 200mL70% precipitation with alcohol.
(3) HPLC detects: sample treatment: 1% aqueous acetic acid 99mL, add the 1ml concentrate, and behind the stirring and evenly mixing, the extract 20 μ L that get different extraction times do the HPLC analysis.Typical curve is: carry out linear regression with peak area (Y) and reference standards concentration (X), getting regression equation is y=2395.3x-1.3591, and R=0.987 goes out qinghaosu concentration according to regression equation calculation.
(4) get 6 parts in the sweet wormwood cured leaf sample of separate sources, repeat 5 times,, after (2) step method is extracted, calculate extraction 1-2h with (3) step with (1); 6-8h; 12-14h; 18-20h; Artemislnin content behind the 24-26h.
Embodiment:
Further illustrate the present invention in the following embodiments, this does not limit the scope of the invention.
Embodiment 1
Get the sweet wormwood of Chongqing fixed star bio-pharmaceuticals Ltd base plantation, take by weighing 100g artemisia leaf dry sample, grind, sample is packed in the filter paper sleeve, sleeve is inserted in the apparatus,Soxhlet's carefully, get 300mL70% ethanol and add in the 600mL flat bottom flask, add several zeolites.The water-bath heating is being extracted 1h respectively, 6h, and 12h, 18h, 24h collects extract, repeats 3 times.Extract with in the hydrochloric acid and after be heated to 70 ℃ after 2% (w/v) activated carbon decolorizing, stir 30min, leave standstill the back to collect supernatant behind the centrifugal 10min of 3000r/min, 10% trichloroacetic acid with 1/5 volume removes albumen again, with the centrifugal removal metaprotein of 3000r/min, supernatant vacuum evaporation concentrates.Residue after concentrating is got the supernatant vacuum after 3000r/min is centrifugal and is concentrated into 30mL with the 200mL70% precipitation with alcohol.Concentrating sample is handled in the following ways: 1% aqueous acetic acid 99mL, add the 1mL extract, and behind the stirring and evenly mixing, be HPLC and analyze.Precision is measured 20ul and is injected liquid chromatograph, the record chromatogram; Chromatographic condition is: chromatographic column: Agilent Eclipse XDB C8 post (5 μ m, 4.6 * 150mm); Moving phase is the second eyeball: acetate buffer solution=25: 75; Temperature: 25 ℃; Flow velocity: 1mL/min; Wavelength: 260nm.Typical curve: carry out linear regression with peak area (Y) and reference substance concentration (X), getting regression equation is y=2395.3x-1.3591, R=0.987.Calculating different extraction time artemislnin content ratio of accounting for dry weight according to formula is: extract 1h, account for 0.113%; Extract 6h, account for 0.261%; Extract 12h, account for 0.415%; Extract 18h, account for 0.614%; Extract 24h, account for 0.619%; Ratio with fully extraction (24h) behind the extraction 1h is 0.183
Get 3 parts of the sweet wormwood cured leafs collected from Qianjiang District of Chongqing peasant household, 3 parts of the sweet wormwood cured leafs that Chongqing sun at tenth of the twelve Earthly Branches peasant household collects extract 1h respectively; 6h; 12h; 18h; Behind the 24h, extract detects with HPLC after purifying.Extract the ratio that artemislnin content behind the 1h accounts for dry weight and be respectively by calculating 6 duplicate samples: 0.123%, 0.117%, 0.128%, 0.210,0.197%, 0.173%, artemislnin content difference is extremely remarkable in the separate sources sweet wormwood.Artemislnin content is respectively 0.180,0.179,0.185 with the ratio that fully extracts (24h) after extracting 1h in the separate sources sweet wormwood cured leaf, 0.178,0.182,0.184, difference is not remarkable, its mean value 0.181, and content is calculated when can be used as qinghaosu and fully extracting.
Embodiment 2
The Soxhlet leaching process is undertaken by embodiment 1, and institute's difference is that HPLC analyzes chromatographic condition and is: chromatographic condition is: chromatographic column: AgilentEclipse XDB C8 post (5 μ m, 4.6 * 150mm); Moving phase is the second eyeball: acetate buffer solution=30: 70; Temperature: 30 ℃; Flow velocity: 1.5mL/min; Wavelength: 260nm.Artemislnin content and the ratio that fully extracts behind the separate sources sweet wormwood cured leaf extraction 1h, difference is not remarkable, and mean value is 0.180.
Claims (3)
1. artemislnin content Fast Detection Technique, this technology comprises (1) qinghaosu Soxhlet rapid extraction and purifying; (2) high performance liquid chromatogram (HPLC) detects.
2. in accordance with the method for claim 1, it is characterized in that with the sweet wormwood cured leaf being that raw material adopts soxhlet extraction to extract qinghaosu, extraction conditions is: 65 ℃-85 ℃ of 70% ethanol water-baths, extraction time is 1-2h, according to the ratio 0.181 of content behind its content and the abundant extraction 24h, extrapolate the artemislnin content of sweet wormwood cured leaf.
3. in accordance with the method for claim 1, it is characterized in that the chromatographic condition that high performance liquid chromatogram (HPLC) detects is: chromatographic column: Agilent EclipseXDB C8 post (5 μ m, 4.6 * 150mm); Moving phase is following prescription choosing base one: second eyeball: acetate buffer solution=25: 75; Second eyeball: acetate buffer solution=30: 70; Second eyeball: acetate buffer solution=25: 75; Temperature: 25-35 ℃; Flow velocity: 1-2mL/min; Wavelength: 260nm.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103159778A (en) * | 2013-03-27 | 2013-06-19 | 中国农业大学 | Hapten, antigen, corresponding antibody and application thereof in detection of artemether |
| CN103197010A (en) * | 2013-04-12 | 2013-07-10 | 张梅 | Technology for rapidly detecting content of artemisinin in artemisia apiacea |
| CN113552274A (en) * | 2021-07-23 | 2021-10-26 | 重庆市中药研究院 | Method for establishing high-performance liquid phase fingerprint spectrum of artemisinin by-product and measuring content of artemisinin by-product |
-
2009
- 2009-10-29 CN CN2009101912583A patent/CN102053121A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103159778A (en) * | 2013-03-27 | 2013-06-19 | 中国农业大学 | Hapten, antigen, corresponding antibody and application thereof in detection of artemether |
| CN103159778B (en) * | 2013-03-27 | 2015-07-22 | 中国农业大学 | Hapten, antigen, corresponding antibody and application thereof in detection of artemether |
| CN103197010A (en) * | 2013-04-12 | 2013-07-10 | 张梅 | Technology for rapidly detecting content of artemisinin in artemisia apiacea |
| CN113552274A (en) * | 2021-07-23 | 2021-10-26 | 重庆市中药研究院 | Method for establishing high-performance liquid phase fingerprint spectrum of artemisinin by-product and measuring content of artemisinin by-product |
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Application publication date: 20110511 |