CN103705559A - Folium artemisiae argyi extract extracted by ethyl acetate and preparation and detection methods and application thereof - Google Patents
Folium artemisiae argyi extract extracted by ethyl acetate and preparation and detection methods and application thereof Download PDFInfo
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- CN103705559A CN103705559A CN201310680696.2A CN201310680696A CN103705559A CN 103705559 A CN103705559 A CN 103705559A CN 201310680696 A CN201310680696 A CN 201310680696A CN 103705559 A CN103705559 A CN 103705559A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 238000001514 detection method Methods 0.000 title claims abstract description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 title claims description 47
- 239000002024 ethyl acetate extract Substances 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 31
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Abstract
本发明涉及艾叶提取物、方法和应用，具体涉及一种艾叶的乙酸乙酯提取物及其制备、检测方法和用途，其有效成分是黄酮类和甾醇类。本发明提供了艾叶提取物在制备抗肿瘤的药物中的应用，并提供了应用超高液相色谱-质谱联机分析技术及结果构建艾叶提取物指纹图谱分析方法。该方法包括艾叶提取物的超高液相色谱-质谱分析条件、色谱峰的特征。色谱组分的质谱提供的化合物结构信息等。本发明公开的指纹图谱及其技术可用于艾叶的品种鉴别及艾叶提取物的质量监控。
The invention relates to the extract, method and application of the leaves of mugwort, in particular to the ethyl acetate extract of the leaves of mugwort, its preparation, detection method and application, and its active ingredients are flavonoids and sterols. The invention provides the application of the Artemisia argyi leaf extract in the preparation of anti-tumor medicines, and provides an analysis method for constructing the fingerprint of the Artemisia argyi leaf extract by using the ultra-high liquid chromatography-mass spectrometry on-line analysis technology and the results. The method includes ultra-high liquid chromatography-mass spectrometry analysis conditions and characteristics of chromatographic peaks of the Artemisia argyi leaf extract. Compound structure information provided by mass spectrometry of chromatographic components, etc. The fingerprint spectrum and the technology disclosed by the invention can be used for the species identification of the mugwort leaf and the quality control of the mugwort leaf extract.
Description
[technical field]
The present invention relates to Folium Artemisiae Argyi extract, methods and applications, be specifically related to ethyl acetate extract and preparation, detection method and the purposes of a kind of Folium Artemisiae Argyi.
[background technology]
Folium Artemisiae Argyi is the dried leaves of feverfew Chinese mugwort Artemisia argyi L é vl.et Vant..There is warming the meridian for stopping bleeding, the effect of dispersing cold for relieving pain, skin pruritus is controlled in clinical, external infertile for haematemesis, epistaxis, few abdomen cold type of pain, coldness and unbalance in meridians, cold womb.Main containing compositions such as volatile oil, flavonoid, triterpenes, tannins.There is the multiple physiologically actives such as hemostasis, anti-bacteria and anti-virus, antitumor, removing free radical, analgesia.
Domestic invention 200810052604.5 discloses with ethyl acetate and alcohol mixture extracts Folium Artemisiae Argyi as solvent; Extracting solution obtains through column chromatography the eluent that eluent obtains with preparative liquid chromatography gradient elution, and mobile phase is water and acetonitrile, collects 40.0 minutes eluents and obtains active component.
Domestic invention 201110131147.0 discloses the extracting method that extracts sesquiterpene lactones compounds Chloroklotzchin from Folium Artemisiae Argyi, first uses ethanol extraction, separated through macroporous adsorbent resin, polyamide and silica gel column chromatography.Finally utilize ethyl alcohol recrystallization, obtained highly purified compound.
Domestic invention 201210117611.5 discloses a kind of Folium Artemisiae Argyi extract, and Folium Artemisiae Argyi is soaked by ethyl acetate, then refluxes, and reflux and finish rear solid-liquid separation, after gained liquid concentration, drying and get final product.For the preparation of anti-hepatic-B virus medicine.
This method is short compared with 200810052604.5 and 201110131147.0 preparation times, method is simple to operate.Its main antineoplastic component is flavonoid and sterols, and 200810052604.5 and 201110131147.0 antineoplastic components are volatile oil composition.201210117611.5 is anti-hepatic-B virus medicine, and this invention is only done color reaction and do not determined its concrete composition.
[summary of the invention]
In order to extract a kind of Folium Artemisiae Argyi effective ingredient that can treat tumor, the invention provides ethyl acetate extract and preparation, detection method and the purposes of a kind of Folium Artemisiae Argyi.
For achieving the above object, design the ethyl acetate extracting method of a kind of Folium Artemisiae Argyi, comprise the following steps:
A. Folium Artemisiae Argyi is soaked 18～36h with petroleum ether, and every 100g Folium Artemisiae Argyi needs the petroleum ether of 300～800ml, and then residue is reclaimed in diafiltration;
B. described residue is concentrated with 70～90% ethanol extraction, and the concentrated solution after concentrating is 1.15～1.20 with respect to the density in ethanol,
C. described concentrated solution is extracted by ethyl acetate, collect the extract of ethyl acetate layer.
Said extracted method also has the scheme of following optimization:
In described step (b), the number of times of ethanol extraction is 2 times, and each extraction time is 2 hours, merges 2 extracting solution and carries out described concentrating.
In described step (b), the concentration of ethanol is 80%.
In step (a), described Folium Artemisiae Argyi is first through pulverizing, and the particle diameter after pulverizing is 250～850 μ m.
In step (a), described immersion, the time needing is 24h.
With said method, extract the Folium Artemisiae Argyi ethyl acetate extract obtaining, main component is flavonoid and sterols.Be specially apigenin, Jaceosidin, luteolin, eupatilin, cupreol and Quercetin.
A pharmaceutical preparation, is comprised of ethyl acetate extract and the pharmaceutically acceptable carrier of above-mentioned Rhizoma Curcumae Longae.
Described preparation is tablet, powder, tablet, capsule or oral liquid.
Detect a method for the ethyl acetate extract of above-mentioned Folium Artemisiae Argyi, with Ultra Performance Liquid Chromatography-electron spray series connection matter instrument, described Folium Artemisiae Argyi extract is analyzed, to obtain the UPLC-Q-TOF-MS collection of illustrative plates of Folium Artemisiae Argyi ethyl acetate extract,
A. mass spectrum condition is: ESI ion source, and positive ion mode detects; Nebulizer N2 pressure is 50psi; Dry gas N2 flow velocity is 15L/min; Mass scanning scope 50～1200; Automatic 3 grades of mass spectrums;
B. chromatographiccondition is: chromatographic column: Waters ACQUITY UPLC
tMbEH C18Column (50mm * 2.1mm.i d., 1.7 μ m); Binary gradient elution, A is 0.1% aqueous formic acid mutually; B is acetonitrile mutually, and flow velocity is 0.3mL/min, and the program of gradient elution is as follows: 90%A phase, reduce gradually A phase ratio, and during to 14min, A phase ratio is 0, keeps 2min, flow velocity: 0.3mL/min; Sample size 3 μ L.
The present invention provides the application of Folium Artemisiae Argyi extract in the medicine of preparation tumor first, and provides application Ultra Performance Liquid Chromatography-mass spectrum on line analytical processing and result to build the method for Folium Artemisiae Argyi extract finger printing.The method comprises the UPLC-MS analysis condition of Folium Artemisiae Argyi extract.The compound object information that the feature of chromatographic peak, the mass spectrum of chromatographic component provide etc.Finger printing disclosed by the invention and technology thereof can be used for the kind discriminating of Folium Artemisiae Argyi and the quality monitoring of Folium Artemisiae Argyi extract.
[accompanying drawing explanation]
Fig. 1 is total ion current figure under Folium Artemisiae Argyi UPLC-Q-TOF/MS positive ion mode.
[specific embodiment]
In order to make object of the present invention, technical scheme and advantage clearer, by reference to the accompanying drawings the present invention is further elaborated.Production equipment in the application is all the common equipment of this area, should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
Embodiment 1:
The ethyl acetate extract of Folium Artemisiae Argyi
By following methods, prepared: Folium Artemisiae Argyi is pulverized, cross 35 mesh sieves and obtain the Folium Artemisiae Argyi granule that particle diameter is 250 μ m, take Folium Artemisiae Argyi granule 500g, with the diafiltration of 2000ml petroleum ether, first with petroleum ether, soak 18h, shoal (percolate is about 2500ml) to percolate color, reclaim residue, volatilize the petroleum ether in residue, with 70% ethanol extraction 2 times, each ethanol consumption is 2500ml, each extraction time is 2 hours, merge extractive liquid, be evaporated to without alcohol taste, relative density is 1.15-1.20, be extracted with ethyl acetate, collect upper layer of extraction liquid, after evaporate to dryness, obtain ethyl acetate extract.
Embodiment 2
The ethyl acetate extract of Folium Artemisiae Argyi
By following methods, prepared: get Folium Artemisiae Argyi and pulverize, cross 20 mesh sieves and obtain the Folium Artemisiae Argyi granule that particle diameter is 850 μ m, take Folium Artemisiae Argyi granule 500g, with 1500ml petroleum ether, soak 24h, obtain residue and percolate (percolate volume is about 2000), residue is volatilized to petroleum ether, concentration is 80% ethanol extraction 2 times, each ethanol consumption is 1500ml, each extraction time is 2 hours, merge extracted twice liquid, be evaporated to relative density 1.15-1.20, obtain ethanol concentrated solution, be extracted with ethyl acetate, obtain upper strata acetic acid ethyl acetate extract, after oven dry, obtain ethyl acetate extract.
Embodiment 3
The ethyl acetate extract of Folium Artemisiae Argyi
By following methods, prepared:: get Folium Artemisiae Argyi and pulverize, cross 30 mesh sieves and obtain the Folium Artemisiae Argyi granule that particle diameter is 450 μ m, Folium Artemisiae Argyi petroleum ether diafiltration, first with 2000ml petroleum ether, soak 36h, obtain residue and percolate (percolate volume is about 2500ml), volatilize the petroleum ether in residue, use 90% ethanol extraction, extract 3 times, each ethanol consumption is 2000ml, extraction time is 3 hours, merge extractive liquid, then be concentrated into without alcohol taste, relative density is 1.15-1.20, be extracted with ethyl acetate, get the acetic acid ethyl acetate extract on upper strata, after oven dry, obtain ethyl acetate extract, after dry, the Folium Artemisiae Argyi extract of gained is brownish black.
The Folium Artemisiae Argyi extract of gained of the present invention has preventive and therapeutic action to tumor.Can, according to conventional formulation technique, Folium Artemisiae Argyi extract of the present invention be prepared into any preparation that is suitable for clinical use, for example powder, tablet.Capsule, oral liquid etc.
Embodiment 4
The granule of the ethyl acetate extract of Folium Artemisiae Argyi
1, form: the ethyl acetate extract of Folium Artemisiae Argyi (the method preparation of embodiment 2) 50g, starch 15g, microcrystalline Cellulose 20g, magnesium stearate 6g.
2, preparation method: take supplementary material according to proportioning, according to the equivalent method mix homogeneously that progressively increases, direct compression.
Embodiment 5
Detection to Folium Artemisiae Argyi anti-tumor drug:
With Ultra Performance Liquid Chromatography-esi-msn, the Folium Artemisiae Argyi ethyl acetate extract of embodiment 1 is analyzed to obtain the finger printing of extract.Its chromatographiccondition is: Waters ACQUITY UPLC
tMbEH C18Column (50mm * 2.1mm.i d., 1.7 μ m) post; Binary gradient elution, A is 0.1% aqueous formic acid mutually, B is acetonitrile flow velocity 0.3mL/min mutually; Sample size 3 μ L.
Preferred chromatographic condition:
Embodiment 6:
Composition detection:
Reagent and sample
Acetonitrile is chromatographically pure, U.S. Fisher company; Ultra-pure water, laboratory ELGA PURELAB Classic-UVF water purification machine, Britain; Other reagent are commercially available domestic analytical pure; Sample is Folium Artemisiae Argyi ethanol extraction.
Sample treatment
Medical material sample is ground into coarse powder, gets 500 grams of coarse powder, first with petroleum ether, soaks diafiltration after 1 day and is extracted into extracting solution (or 5 times of medical material amounts) of light color, reclaims petroleum ether, obtains ligroin extraction; Residue is flung to petroleum ether, moistening with 80% ethanol after, then with 3 times of twice of 80% alcohol reflux of amount, merge extractive liquid,, decompression recycling ethanol, to without alcohol taste, extracts 2 times by equal-volume ethyl acetate, combined ethyl acetate extract, reclaims ethyl acetate to dry, obtains acetic acid ethyl ester extract; Mother solution is concentrated into dry, obtains ethanol extraction; Residue volatilizes ethanol, with 3 times of water gagings, boils and carries 2 times, merges aqueous extract, is concentrated into dryly, obtains aqueous extract.
Instrument and condition
U.S. Waters Acquity
tMuPLC/Q-TOF Premier; MassL-ynxV4.1 work station.
Chromatographic condition is: 90%A phase, reduce gradually A phase ratio, and during to 14min, A phase ratio is 0, keeps 2min, flow velocity: 0.3mL/min; Sample size 3 μ L.
Mass spectrum condition: ESI ion source, positive ion mode detects; Nebulizer N2 pressure 50psi; Dry gas N2, flow velocity 15L/min; Mass scanning scope 50--1200; Automatic 3 grades of mass spectrums.(automatically selecting the strongest ion to carry out induction cracking in source).
Results and analysis
UPLC-Q-TOF-MS(﹢) secondary and three grades of mass spectrums of leading ion in the ion that analysis result and finger printing information chromatographic component one-level mass spectrum thereof comprise and one-level mass spectrum.
The peak component finger print information of the total ions chromatogram of the UPLC-Q-TOF of Folium Artemisiae Argyi extract-MS(﹢ in Fig. 1) analyzing is as shown in the table:
Embodiment 7
Activity determination method:
By the Folium Artemisiae Argyi in embodiment 1 for ethyl acetate extract homogeneous phase time discrimination fluorescence (HTRF) method survey the activity of enzyme, start reaction after adding ATP, kinases makes substrate generation phosphorylation reaction, and phosphate radical has been connected on biotin labeled substrate.In this process, EDTA has stopped the carrying out of reaction, have on the phosphate radical of the anti-phosphorylated tyrosine antibody of europium rubidium marking near substrate, the allophycocyanin of labelling XL665 is combined on the biotin labeling of substrate, in process adjacent to each other, there is resonance energy transfer and at 665nm place, produce fluorescence in two fluorophors, at 620nm place, there is fluorescence in free TK antibody (labelling europium ion), this signal can be used as background signal, and free SA-XL665 only produces of short duration fluorescence, by delaying after adding terminator detection time, within 1 hour, detect again, can be ignored.The signal value that final instrument provides is calculated by following formula: Ratio=(665nm/620nm) * 10
4.
In reaction vessel, every hole adds EGFR kinase solution compound Large-scale Screening to carry out in 384 orifice plates, with PP-550MS pipettor transfer liquid, in reaction vessel, every hole adds EGFR kinase solution 2 μ l, substrate solution 2 μ l, buffer or compound to be sieved 4 μ l, ATP2 μ l.React 1 hour.Every hole adds TK-Ab5 μ l, SA-XL6655 μ l, incubated at room 1 hour.Utilize Beckman Coulter detection platform HTRF module to detect.The non-selective inhibitor of EGFR kinases is Staurosporine(spore rhzomorph), with the reliability of the method for inspection.
Sample screening
Primary dcreening operation Folium Artemisiae Argyi ethyl acetate extract, carries out multiple sieve to high activity sample after calculating suppression ratio.While sieving again, to dilute be successively 5 concentration to each sample, and experiment in triplicate, is respectively 50,5,0.5,0.05,0.005 μ gmL
-1.According to measurement result, calculate IC
50.
Result
The rate of being inhibited is greater than 80% Folium Artemisiae Argyi extract and carries out multiple sieve, is positive, and dilutes successively 5 concentration, calculates IC
50.From following table, can find out: four positions of Folium Artemisiae Argyi have EGFR kinase inhibiting activity in various degree.It is the strongest that its ethyl acetate extract suppresses activity, is being 50 μ gmL at final concentration
-1time, suppression ratio is respectively 18.36%, 26.17%, and 82.28%, 15.29%.Folium Artemisiae Argyi ethyl acetate extract IC
50be 8.848 μ gmL
-1, other three positions do not detect.
Claims (10)
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Cited By (5)
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| CN104297377A (en) * | 2014-10-24 | 2015-01-21 | 曲阜师范大学 | Detection and analysis method of phytosterol |
| CN104478842A (en) * | 2014-12-24 | 2015-04-01 | 陕西嘉禾植物化工有限责任公司 | Method for extracting jaceosidin and eupatilin from folium artemisiaeargyi |
| CN108152416A (en) * | 2018-01-03 | 2018-06-12 | 艾江山健康科技蕲春有限公司 | The finger print measuring method of active constituent in Chinese mugwort acid extract |
| CN111588741A (en) * | 2020-05-13 | 2020-08-28 | 扬州大学 | Preparation method and application of folium artemisiae argyi extract |
| CN114136737A (en) * | 2021-12-08 | 2022-03-04 | 河南中医药大学 | A kind of microsection method for observing the characteristics of the lower surface of Artemisia argyi |
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| CN102600225A (en) * | 2012-04-20 | 2012-07-25 | 河南省医药科学研究院 | Wormwood leaf extract and application thereof to preparation of anti-hepatitis B virus medicines |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104297377A (en) * | 2014-10-24 | 2015-01-21 | 曲阜师范大学 | Detection and analysis method of phytosterol |
| CN104478842A (en) * | 2014-12-24 | 2015-04-01 | 陕西嘉禾植物化工有限责任公司 | Method for extracting jaceosidin and eupatilin from folium artemisiaeargyi |
| CN108152416A (en) * | 2018-01-03 | 2018-06-12 | 艾江山健康科技蕲春有限公司 | The finger print measuring method of active constituent in Chinese mugwort acid extract |
| CN111588741A (en) * | 2020-05-13 | 2020-08-28 | 扬州大学 | Preparation method and application of folium artemisiae argyi extract |
| CN114136737A (en) * | 2021-12-08 | 2022-03-04 | 河南中医药大学 | A kind of microsection method for observing the characteristics of the lower surface of Artemisia argyi |
| CN114136737B (en) * | 2021-12-08 | 2024-02-23 | 河南中医药大学 | Microscopic tabletting method for observing lower surface characteristics of mugwort leaves |
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