CN109917065A - The identification method of liposoluble ingredient and its application in a kind of green propolis - Google Patents
The identification method of liposoluble ingredient and its application in a kind of green propolis Download PDFInfo
- Publication number
- CN109917065A CN109917065A CN201910302237.8A CN201910302237A CN109917065A CN 109917065 A CN109917065 A CN 109917065A CN 201910302237 A CN201910302237 A CN 201910302237A CN 109917065 A CN109917065 A CN 109917065A
- Authority
- CN
- China
- Prior art keywords
- green propolis
- green
- propolis
- esi
- identification method
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Jellies, Jams, And Syrups (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a kind of identification method of liposoluble ingredient in green propolis and its applications, green propolis or green propolis extracted with alcohol methanol ultrasonic extraction are obtained into analysis test liquid through micro-pore-film filtration, it is measured using UPLC-ESI-qTOF-MS, determines liposoluble ingredient in green propolis.The present invention establishes the identification method of liposoluble ingredient in green propolis for the first time, sample is with methanol ultrasonic extraction, and it is measured using ultrahigh pressure liquid phase chromatograph-mass spectrometer coupling method (UPLC-ESI-qTOF-MS), negative ions mode under ESI ion source can be with the ingredient of phenolic acid class in accurate determining propolis, it is fast with detection time, sensitivity and accuracy is preferable, stability is strong, it is easy to operate, the advantages that easy to implement, provides a kind of new method for the control of the quality of green propolis and green propolis product.
Description
Technical field
The invention belongs to the technical field of quality detection of green propolis, and in particular to the mirror of liposoluble ingredient in a kind of green propolis
Determine method and its application.
Background technique
The green propolis type of Brazil is more, has preferable biological activity, is the propolis that quality is best in the world, so as to cause
The extensive concern of domestic and international researcher.Current green propolis is also applied to the fields such as medicine, food, cosmetics more and more widely,
Principal item has pure green propolis, green bee glue powder etc..By the strain and locality shadows such as geographical environment and process of plant
It rings, the complex chemical composition of propolis.Green propolis in the market and products thereof lacks unified quality control standard, and quality is caused to be supervised
Pipe portion door and production and operation enterprise can not the quality to green propolis effectively supervised and controlled.
Once studies have reported that determining green Flavonoids in Propolis constituents, including Galangin, rutin, myricetin, Kaempferol,
The ingredients such as chrysin, using the content of flavones ingredient as quality control index.But the main component of green propolis is phenolic acid compound, yellow
The content of ketone compounds is lower, and therefore, the quality that propolis is controlled only by the content of general flavone is also far from enough.
105929074 A of CN discloses a kind of green propolis method of quality control of Brazil, and high performance liquid chromatography is used to survey
The content of 6 evaluation index ingredients is determined.But its detection time is up to 90min, low efficiency, and solvent consumption is big, uneconomical.Cause
This, develops one kind and precisely, quickly identifies that the method for phenolic acid compound in green propolis seems very necessary, to be formed with
The green propolis method of quality control of effect, provides reference frame for green propolis product in the market.
Summary of the invention
In consideration of it, this method can the purpose of the present invention is to provide a kind of identification method of liposoluble ingredient in green propolis
Quickly and accurately to identify the liposoluble ingredient in propolis, one kind is provided for the control of the quality of green propolis and green propolis product
New method.
The present invention is achieved through the following technical solutions:
The present invention provides a kind of identification methods of liposoluble ingredient in green propolis, and green propolis or green propolis extracted with alcohol are used
Methanol ultrasonic extraction is obtained analysis test liquid, is measured, is determined green using UPLC-ESI-qTOF-MS through micro-pore-film filtration
A variety of liposoluble ingredients in propolis.
Preferably, the mass volume ratio of the green propolis or green propolis extracted with alcohol and methanol are as follows: 0.1-0.5g:
10ml。
Preferably, the time of the ultrasonic extraction is 20-60min.
The present invention will analyze test liquid using ultrahigh pressure liquid phase chromatograph-mass spectrometer coupling method (UPLC-ESI-qTOF-MS) into
Row measures, and the negative ions mode under ESI ion source can be with a variety of liposoluble ingredients in the green propolis of accurate determination, preferably
, the UPLC-ESI-qTOF-MS chromatographic condition are as follows:
Using octadecylsilane chemically bonded silica as filler；Flow velocity is 0.2-0.5mL/min；Column temperature: 30 DEG C；Sample volume: 2.00 μ
L；Mobile phase A: 0.1-0.3% aqueous formic acid；Mobile phase B: methanol；Gradient elution program is as shown in the table:
Preferably, the UPLC-ESI-qTOF-MS Mass Spectrometry Conditions are as follows: electric spray ion source, negative ions mode, atomization gas are
Nitrogen, Mass Spectrometer Method range: MS/100 ~ 1700；MS/MS:50 ~ 1000；Dry air pressure: 30-50PSI, 320 DEG C of temperature, flow velocity
8-11L/min, empty tubule voltage: 3000-4000V；Taper hole gas (N2) flow velocity: 10-15L/min, temperature: 350 DEG C；Ion source temperature
Degree: 100-250 DEG C.
The method of the present invention obtains the total ion current figure of UPLC-ESI-qTOF-MS according to above-mentioned chromatography and mass spectral analysis condition
With the first mass spectrometric figure of each ingredient, through a variety of liposoluble ingredients in the determining green propolis of analysis be respectively as follows: chlorogenic acid, caffeic acid,
3,4-Dicaffeoylquinic acid, 3,5-Dicaffeoylquinic acid, 4,5-Dicaffeoylquinic acid, phenethyl caffeate, Aunar woods C, baccharine.
Another object of the present invention is to provide the identification methods of liposoluble ingredient in above-mentioned green propolis in green propolis matter
Application in amount detection.
Compared with prior art, the present invention having the following beneficial effects:
The present invention establishes the identification method of liposoluble ingredient in green propolis for the first time, and sample is used super with methanol ultrasonic extraction
High pressure liquid chromatography-mass spectrometry combination method (UPLC-ESI-qTOF-MS) is measured, the negative ions mode under ESI ion source
Can be with a variety of liposoluble ingredients in accurate determining propolis, this method has detection time fast, sensitivity and accuracy preferably,
The advantages that stability is strong, easy to operate, easy to implement provides a kind of new for the control of the quality of green propolis and green propolis product
Method.
Detailed description of the invention
Fig. 1 is green propolis sample UPLC-ESI-qTOF-MS total ion current figure；
Fig. 2 is the first mass spectrometric figure (ESI+&ESI-) of chlorogenic acid；
Fig. 3 is caffeinic first mass spectrometric figure (ESI+&ESI-)；
Fig. 4 is the first mass spectrometric figure (ESI+&ESI-) of 3,4-Dicaffeoylquinic acid；
Fig. 5 is the first mass spectrometric figure (ESI+&ESI-) of 3,5-Dicaffeoylquinic acid；
Fig. 6 is the first mass spectrometric figure (ESI+&ESI-) of 4,5-Dicaffeoylquinic acid；
Fig. 7 is the first mass spectrometric figure (ESI+&ESI-) of phenethyl caffeate；
Fig. 8 is the first mass spectrometric figure (ESI+&ESI-) of Aunar woods C；
Fig. 9 is the first mass spectrometric figure (ESI+&ESI-) of baccharine.
Specific embodiment
Further illustrate that the present invention, following embodiment are the specific embodiment party of the present invention below by specific embodiment
Formula, but embodiments of the present invention are not limited by following embodiments, it is other any without departing from Spirit Essence of the invention
With changes, modifications, substitutions, combinations, simplifications made under principle, equivalent substitute mode should be, be included in of the invention
Within protection scope.
Raw material of the present invention derives from commercially available.
One, material and method
1.1 instrument
Ten a ten thousandth electronic balances；UPLC-ESI-qTOF-MS LC-MS instrument；Ultrasonic washing instrument.
1.2 reagent
Methanol: chromatographically pure, Merck & Co., Inc.；Dehydrated alcohol: analysis is pure；Formic acid: analysis is pure；C18 chromatographic column: Agilent.
Two, method
The preparation of 2.1 test samples
Green propolis sample 0.3g is weighed, is placed in 10mL volumetric flask, ultrasound 30min after methanol constant volume is added, 20min is stood, takes
Supernatant；It crosses 0.45 μm of filter membrane and analyzes test liquid to get to UPLC-ESI-qTOF-MS.
2.2UPLC-ESI-qTOF-MS chromatographic condition
Chromatographic column Agilent SB-C18 column (2.1 × 100 mm, 1.8 μm)；Flow velocity is 0.3 mL/min；Column temperature: 30 DEG C；
Sample volume: 2.00 μ L；Mobile phase A: 0.1% aqueous formic acid；Mobile phase B: methanol.Gradient elution program is as shown in the table:
2.3UPLC-ESI-qTOF-MS Mass Spectrometry Conditions
Electric spray ion source (Dual AJS ESI), negative ions mode, atomization gas are nitrogen, Mass Spectrometer Method range: MS/100 ~
1700；MS/MS:50 ~ 1000；Dry air pressure: 35PSI, 320 DEG C of temperature, flow velocity 8L/min, empty tubule voltage: 3500V；Taper hole
Gas (N2) flow velocity: 11L/min, temperature: 350 DEG C；Ion source temperature: 150 DEG C.
Liposoluble ingredient UPLC-ESI-qTOF-MS qualification result in 2.4 green propolis
By above-mentioned chromatography and mass spectral analysis condition, the total ion current figure and first mass spectrometric figure of UPLC-ESI-qTOF-MS is obtained, such as
Shown in Fig. 1-9.By analysis under relative retention time, positive and negative ion ionization mode molecular ion peak accurate molecular weight, and
The data such as the molecular weight of fragment ion and cleavage of mass spectrum rule parse different chromatographic peaks and represent different phenolic acid compositions, obtain
The qualification result of main liposoluble ingredient is as shown in the table:
Three, methodological study:
1, the selection of mobile phase and elution requirement
During the experiment, acetonitrile-water, acetonitrile-water (containing 0.1% formic acid), acetonitrile-water (containing 0.1% acetic acid), methanol-are compared
Water, methanol-water (containing 0.1% formic acid) or methanol -- separation appraisal when water (containing 0.1% acetic acid) is mobile phase, discovery 0.1%
The addition of formic acid or acetic acid, can be obviously improved chromatographic peak hangover situation, a small amount of formic acid and acetic acid during electrospray ionisation also
The response signal of mass spectra peak can be remarkably reinforced, but its ms fragment of the addition of acetic acid parses opposite formic acid and wants complicated；In addition,
In comparison, methanol is also more preferable than the more cheap of acetonitrile, safety.So (being contained in present invention selection using methanol-water
0.1% formic acid) it is used as mobile phase.
Equally, during the experiment, it using water (containing 0.1% formic acid) (A)-methanol (B) as mobile phase, compares not on year-on-year basis
The mobile phase isocratic elution and several different gradient elution programs of example.
Isocratic elution program (I): the min of 0 min ~ 40, water (containing 0.1% formic acid, A, 60%)-methanol (B, 40%)；It is isocratic to wash
De- program (): the min of 0 min ~ 36, water (containing 0.1% formic acid, A, 40%)-methanol (B, 60%).
Gradient elution program (): 0 min ~ 4 min, 40% → 60% (B)； 4 min~20 min, 60% →
80% (B)；20 min~25 min, 80% → 90% (B)；25 min~30 min, 90% → 90% (B).
Gradient elution program (): 0 ~ 10 min, 30 % → 45 % (B)；10~20 min, 45 %→55 % (B)；
20~30 min, 55 %→75% (B)；30 ~ 36 min, 75% → 90 % (B);36 ~ 40 min, 90 % → 95 % (B);
40~45 min, 95 % (B) 。
Gradient elution program (): 0 ~ 11 min, 25 % → 45 % (B)；11~22.5 min, 45 %→55 %
(B)；22.5~29min, 55 %→70% (B)；29 ~ 37.5 min, 70% → 80 % (B);37.5 ~ 40 min, 80 % → 95
% (B); 40~44 min, 95 % (B) ；44~46.5 min, 95 % -25%(B)；46.5~50 min, 25%(B)；.
The study found that green propolis or green propolis extracted with alcohol complicated composition, flavones phenolic acid class composition therein is through flowing
The separating effect of equality elution is poor, and gradient elution separation situation is preferable.And for gradient elution analysis, with mobile phase
The ratio variation of middle first alcohol and water (containing 0.1% formic acid) has substantial connection, finds through analysis and research, selection gradient elution program (
), separating effect is optimal.
2, sample solution preparation method is investigated
Link is prepared in test solution, the Different solutions such as methanol, ethyl alcohol, acetonitrile, pure water has been investigated and above-mentioned solvent is different
The mixed solvent of ratio, discovery methanol, ethyl alcohol, acetonitrile can be dissolved for sample well, and pure water is not suitable for.It is more different again
When the aqueous solution of the methanol of concentration, ethyl alcohol and acetonitrile, it is found that the pure and mild acetonitrile of higher proportion also can be compared with good dissolving for sample, still
It is wherein best with the effect of pure methanol and pure acetonitrile.In view of methanol liquid matter connection more cheap compared to ethyl alcohol and safe and subsequent
It is methanol-water solution with analysis mobile phase used, so final choice methanol of the present invention prepares solvent as test sample.
Claims (7)
1. the identification method of liposoluble ingredient in a kind of green propolis, which is characterized in that by green propolis or green propolis extracted with alcohol
Analysis test liquid is obtained, is measured using UPLC-ESI-qTOF-MS, is determined through micro-pore-film filtration with methanol ultrasonic extraction
A variety of liposoluble ingredients in green propolis.
2. the identification method of liposoluble ingredient in a kind of green propolis according to claim 1, which is characterized in that the green bee
The mass volume ratio of glue or green propolis extracted with alcohol and methanol are as follows: 0.1-0.5g:10ml.
3. the identification method of liposoluble ingredient in a kind of green propolis according to claim 1, which is characterized in that the ultrasound
The time of extraction is 20-60min.
4. the identification method of liposoluble ingredient in a kind of green propolis according to claim 1, which is characterized in that described
UPLC-ESI-qTOF-MS chromatographic condition are as follows:
Using octadecylsilane chemically bonded silica as filler；Flow velocity is 0.2-0.5mL/min；Column temperature: 30 DEG C；Sample volume: 2.00 μ
L；Mobile phase A: 0.1-0.3% aqueous formic acid；Mobile phase B: methanol；Gradient elution program is as shown in the table:
。
5. the identification method of liposoluble ingredient in a kind of green propolis according to claim 1, which is characterized in that described
UPLC-ESI-qTOF-MS Mass Spectrometry Conditions are as follows: electric spray ion source, negative ions mode, atomization gas are nitrogen, Mass Spectrometer Method model
It encloses: MS/100 ~ 1700；MS/MS:50 ~ 1000；Dry air pressure: 30-50PSI, 320 DEG C of temperature, flow velocity 8-11L/min, empty tubule
Voltage: 3000-4000V；Taper hole gas velocity: 10-15L/min, 350 DEG C of temperature；Ion source temperature: 100-250 DEG C.
6. the identification method of liposoluble ingredient in a kind of green propolis according to claim 1, which is characterized in that is determined is green
A variety of liposoluble ingredients are respectively as follows: chlorogenic acid, caffeic acid, 3,4-Dicaffeoylquinic acid, 3,5-Dicaffeoylquinic acid, 4,5-Dicaffeoylquinic acid, caffeic acid in propolis
Phenethyl ester, Aunar woods C, baccharine.
7. the identification method of liposoluble ingredient is in green propolis quality in a kind of green propolis according to claim 1-6
Application in detection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910302237.8A CN109917065A (en) | 2019-04-16 | 2019-04-16 | The identification method of liposoluble ingredient and its application in a kind of green propolis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910302237.8A CN109917065A (en) | 2019-04-16 | 2019-04-16 | The identification method of liposoluble ingredient and its application in a kind of green propolis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109917065A true CN109917065A (en) | 2019-06-21 |
Family
ID=66977436
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910302237.8A Pending CN109917065A (en) | 2019-04-16 | 2019-04-16 | The identification method of liposoluble ingredient and its application in a kind of green propolis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109917065A (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009184929A (en) * | 2008-02-01 | 2009-08-20 | Api Co Ltd | Supercritical extract, its manufacturing method, its detection method, and food and drink, cosmetic and pharmaceutical containing the same |
| TW201014649A (en) * | 2008-10-14 | 2010-04-16 | Nat Univ Chung Hsing | Purification of bioactive compounds recovered from propolis by using supercritical fluids crystallization |
| CN103175922A (en) * | 2013-03-08 | 2013-06-26 | 浙江大学 | Baccharis dracunculifolia propolis quality control method |
| CN103257201A (en) * | 2013-04-12 | 2013-08-21 | 杭州蜂之语蜂业股份有限公司 | Method for simultaneous determination of caffeic acid and 8 flavonoids in propolis by using high performance liquid chromatography |
| CN103267823A (en) * | 2013-05-27 | 2013-08-28 | 杭州蜂之语蜂业股份有限公司 | Method for measuring content of Artepillin C in propolis by high performance liquid chromatography method |
| CN103592383A (en) * | 2013-11-06 | 2014-02-19 | 浙江大学 | Quality control method and application of baccharis type propolis |
| CN105929074A (en) * | 2016-04-28 | 2016-09-07 | 浙江大学 | Brazilian green propolis quality control method |
-
2019
- 2019-04-16 CN CN201910302237.8A patent/CN109917065A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009184929A (en) * | 2008-02-01 | 2009-08-20 | Api Co Ltd | Supercritical extract, its manufacturing method, its detection method, and food and drink, cosmetic and pharmaceutical containing the same |
| TW201014649A (en) * | 2008-10-14 | 2010-04-16 | Nat Univ Chung Hsing | Purification of bioactive compounds recovered from propolis by using supercritical fluids crystallization |
| CN103175922A (en) * | 2013-03-08 | 2013-06-26 | 浙江大学 | Baccharis dracunculifolia propolis quality control method |
| CN103257201A (en) * | 2013-04-12 | 2013-08-21 | 杭州蜂之语蜂业股份有限公司 | Method for simultaneous determination of caffeic acid and 8 flavonoids in propolis by using high performance liquid chromatography |
| CN103267823A (en) * | 2013-05-27 | 2013-08-28 | 杭州蜂之语蜂业股份有限公司 | Method for measuring content of Artepillin C in propolis by high performance liquid chromatography method |
| CN103592383A (en) * | 2013-11-06 | 2014-02-19 | 浙江大学 | Quality control method and application of baccharis type propolis |
| CN105929074A (en) * | 2016-04-28 | 2016-09-07 | 浙江大学 | Brazilian green propolis quality control method |
Non-Patent Citations (3)
| Title |
|---|
| DEEPAK KASOTE ET AL: "Chemical profiling and chemometric analysis of South African propolis", 《BIOCHEMICAL SYSTEMATICS AND ECOLOGY》 * |
| EWELINA SZLISZKA,ET AL: "Chemical Composition and Anti-Inflammatory Effect of Ethanolic Extract of Brazilian Green Propolis on Activated J774A.1 Macrophages", 《EVIDENCE-BASED COMPLEMENTARY AND ALTERNATIVE MEDICINE》 * |
| JULIANNA KARLA SANTANA ANDRADE ET AL: "Development and characterization of microencapsules containing spray dried powder obtained from Brazilian brown, green and red propolis", 《FOOD RESEARCH INTERNATIONAL》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Natić et al. | Analysis and characterisation of phytochemicals in mulberry (Morus alba L.) fruits grown in Vojvodina, North Serbia | |
| Julianti et al. | Quantification of the antiplasmodial alkaloid carpaine in papaya (Carica papaya) leaves | |
| CN102735784A (en) | Method for simultaneously determining one hundred pesticide residuals in traditional Chinese medicine through ultrahigh performance liquid chromatography-tandem quadrupole mass spectrum | |
| CN104614466B (en) | The assay method of antiseptic in tobacco juice for electronic smoke | |
| Liu et al. | Rapid analysis of 27 components of Isodon serra by LC–ESI-MS–MS | |
| CN103792278B (en) | Electrospray extraction ionization-mass spectrum (EESI-MS) rapid detection method for alkaloid in lotus seeds | |
| CN105403630A (en) | Method for detecting concentration of nine compound coloring agents in auxiliary materials for reconstituted tobacco | |
| CN106596792B (en) | A kind of method of aromatic ester content of material in detection white wine | |
| CN106814155B (en) | The chiral analysis of nicotine closes phase chromatographic tandem mass spectrography in a kind of tomato | |
| CN107064362B (en) | The measurement of parabens substance in buccal cigarette | |
| Zhao et al. | Simultaneous determination of ten flavonoids from Viscum coloratum grown on different host species and different sources by LC-MS | |
| Sun et al. | Simultaneous analysis of 11 main active components in C irsium setosum based on HPLC‐ESI‐MS/MS and combined with statistical methods | |
| Du et al. | Simultaneous qualitative and quantitative analysis of 28 components in Isodon rubescens by HPLC‐ESI‐MS/MS | |
| Marinho et al. | A validated method for the simultaneous quantitation of bioactive alkaloid markers in the leaf ethanolic extract of Cissampelos sympodialis Eichl.: a phenological variation study | |
| Xiong et al. | Study on phenolic acids of Lonicerae japonicae Flos based on ultrahigh performance liquid chromatography‐tandem mass spectrometry combined with multivariate statistical analysis | |
| CN101852786A (en) | Method for detecting sulfur dioxide content in shitake mushrooms | |
| CN112710765A (en) | Fingerprint detection method of gardenia medicinal material and application thereof | |
| CN112114079B (en) | Method for simultaneously detecting 9 chemical components in quisqualis indica | |
| Xu et al. | Qualitative and quantitative determination of nine main active constituents in Pulsatilla cernua by high‐performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry | |
| CN106404956B (en) | High performance liquid chromatography-tandem mass internal standard method detects the method and its application of four kinds of organic acids in grape wine and/or fruit wine simultaneously | |
| CN109917065A (en) | The identification method of liposoluble ingredient and its application in a kind of green propolis | |
| CN106404507A (en) | Leaf vegetable gibberellins residue detection sample pretreatment method and detection method | |
| CN105954434A (en) | Method for detecting phenols spice | |
| CN108535387B (en) | Combined phase chromatography tandem mass spectrometry determination method of NNK and NNN in tobacco | |
| CN103808822A (en) | LC-QTOF (Liquid Chromatography-Quadrupole Time Of Flight) analysis method for distinguishing resveratrol of different resources |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190621 |
|
| RJ01 | Rejection of invention patent application after publication |