CN101620208A - Identification method for propolis and poplar gum - Google Patents
Identification method for propolis and poplar gum Download PDFInfo
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- CN101620208A CN101620208A CN200910101428A CN200910101428A CN101620208A CN 101620208 A CN101620208 A CN 101620208A CN 200910101428 A CN200910101428 A CN 200910101428A CN 200910101428 A CN200910101428 A CN 200910101428A CN 101620208 A CN101620208 A CN 101620208A
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Abstract
The invention provides an identification method for propolis and poplar gum, which respectively identifies fingerprints of the propolis, the poplar gum and a mixture doped with different proportions of propolis by a reverse phase high-performance liquid chromatograph, and judges the truth of the propolis by the comparison of the fingerprints so as to control the quality of the propolis. The invention controls the quality of the propolis by using an HPLC method, has favorable separation effect, high sensitivity and high feasibility; the propolis and the poplar gum in the method respectively have obvious characteristic peaks, are free from mutually intervening and are easy to identify, thereby mass spectrum, standard products and the like are not needed, and the cost is reduced. In addition, the method has high sensitivity, is easy to grasp, can be used for identifying the propolis from the poplar gum and can simultaneously judge whether the propolis is doped with the poplar gum or not.
Description
Technical field
The invention belongs to food, drug quality control method, relate generally to a kind of propolis method of quality control, this method is to utilize liquid chromatography to carry out the discriminating of propolis and yang gum.
Background technology
Propolis is that secretion such as honeybee herborization resin mixes the tackiness material that forms with secretion such as its mandibular gland, wax glands.Various biological effects such as propolis has the prevention wound infection, it is immune, antitumor to regulate, reducing blood lipid, hypoglycemic and resisting pathogenic microbes now have been widely used in healthcare industry, and have been put into Pharmacopoeia of People's Republic of China in 2005.Along with the raising of people's living standard, the domestic and international market increases day by day to the demand of propolis.
Because propolis output is few, price is higher relatively, and some lawless persons palm off propolis extract with the gemma populi extract, and these yang gums are at color, smell, all quite similar with true propolis in shape.The production cost of this false propolis will be significantly less than true propolis, and with its propolis product of making, natural cost is just low a lot.Although false propolis has at aspects such as general flavones and true propolis similarity, but some physiological activator that it does not have honeybee to be added in processing propolis process, and the raw material of false propolis and process can be brought a lot of unsafe factors, therefore, the product of making raw material with yang gum of poor quality comes into the market, and brings endless trouble.And main test item is in existing propolis national standard of China and the industry standard: state, smell, color, ethanol extract content, beeswax, oxidization time, general flavone content etc.These indexs can't detect the propolis that is mixed with yang gum, more can't differentiate the propolis and the yang gum of purifying.Owing to lack the method that corresponding true and false propolis is differentiated, quality monitoring department and processing, trading enterprise can't effectively supervise and control in the propolis production and the process of circulation, false propolis is mixed the spurious with the genuine, and the value of true propolis can't be embodied, and consumer's rights and interests can't be protected.Therefore, how to identify efficiently and accurately that the quality of propolis and the true and false have important application.
Summary of the invention
The object of the present invention is to provide the discrimination method of a kind of propolis and yang gum, the liquid phase chromatography of this method by precise and high efficiency is to being that the propolis and the yang gum of main glue source plant production differentiated with willow, with control propolis quality.
The objective of the invention is to realize by following scheme:
(1) with propolis with 95% ethanol ultrasonic extraction, centrifugal, filter, obtain propolis and extract solution;
(2) remove ethanol in the propolis ethanol extract, add chloroform extraction, remove the chloroform phase, repeatedly several times, stay water, wave the chloroform of clean aqueous phase, use the moving phase constant volume with separating funnel;
(3) analyze with rp-hplc, obtain the chromatogram of propolis;
(4) repeat the method for above-mentioned (1), (2), (3), obtain yang gum and be mixed with the chromatogram of the propolis of yang gum;
(5) with pure propolis, yang gum compares with the chromatogram that is mixed with the propolis of yang gum, thereby judges the true and false of propolis.
The condition of liquid chromatograph of the present invention is: flow velocity is 0.5～1.0mL/min, detecting wavelength is 213～220nm or 260～270nm, sample size is 2～10 μ L, chromatographic column is Agilent Eclipse XDB-C18 (4.6 * 250mm, 5 μ m) or suitable chromatographic column, column temperature is 25～30 ℃.
Moving phase of the present invention is methyl alcohol: 0.5%H
3PO
4=14: 86 (volumetric ratios).
Another object of the present invention provides the application of discrimination method in propolis and yang gum discriminating of this propolis and yang gum.
Two tangible peaks are arranged in the propolis finger-print of the present invention, and do not contain these two peaks under the same terms in the yang gum, the priority that occurs according to these two peaks is numbered No. 1 peak and No. 2 peaks respectively; Two tangible peaks are arranged in the yang gum finger-print, do not contain this two peaks under the same conditions in the propolis, be numbered No. 3 peaks and No. 4 peaks respectively.According to said method, measured propolis, yang gum respectively and mixed the finger-print of the propolis of different proportion.By the chromatogram that obtains as can be known, contain in the propolis finger-print No. 1 and No. 2 characteristic peaks, do not contain No. 3 and No. 4 peaks; Contain in the yang gum finger-print No. 3 and No. 4 peaks, do not contain No. 1 and No. 2 peaks; Contain the peak simultaneously 1,2,3 and No. 4 and be mixed with in the propolis of 10～30% yang gums.
The present invention is that the propolis sample of main glue source plant production is verified repeatedly through what pick up from different regions with willow, prove this method good separating effect, highly sensitive, feasibility is strong, therefore, can differentiate propolis and yang gum, can judge whether mix yang gum in the propolis simultaneously.
The inventive method utilization HPLC method is carried out the propolis quality control, and characteristic peak is obvious separately with yang gum for propolis related in the method, each other not interference, easily differentiate, therefore need not use mass spectrum, standard items etc. to prove conclusively, reduced cost, and the inventive method is highly sensitive, easily grasps.
Description of drawings
Fig. 1 is the chromatogram of example 1 propolis.
Fig. 2 is the chromatogram of example 1 yang gum.
Fig. 3 mixes the chromatogram of 10% yang gum for example 1.
Fig. 4 mixes the chromatogram of 20% yang gum for example 1.
Fig. 5 mixes the chromatogram of 30% yang gum for example 1.
Fig. 6 is the chromatogram of example 2 propolis.
Fig. 7 is the chromatogram of example 2 yang gums.
Fig. 8 is the chromatogram of example 3 propolis.
Fig. 9 is the chromatogram of example 3 yang gums.
Figure 10 is the chromatogram of example 4 propolis.
Figure 11 is the chromatogram of example 4 yang gums.
Figure 12 is the chromatogram of example 5 propolis.
Figure 13 is the chromatogram of example 5 yang gums.
Specific embodiments
The present invention is further described in conjunction with the accompanying drawings and embodiments.
Embodiment 1:
Get propolis 10g, centrifugal with 80mL 95% ethanol ultrasonic dissolution, filter, obtain propolis solution, remove ethanol with boulton process, add low amounts of water, use chloroform extraction, remove the residual chloroform of water-soluble part, to 25mL, mixing with 0.22 μ m membrane filtration, is analyzed with HPLC with the moving phase constant volume.Adopt the chromatographic column of AgilentXDB-C18 (4.6 * 250mm, 5 μ m), flow velocity is adjusted to 0.6mL/min, detects wavelength 220nm, 30 ℃ of column temperatures, sample size 5 μ L.Referring to Fig. 1.No. 1 peak and No. 2 peaks are the propolis characteristic peak among the figure.
Get yang gum 2.5g, with 80mL 95% ethanol ultrasonic dissolution, centrifugal, filter, obtain yang gum solution, remove ethanol, add low amounts of water with boulton process, use chloroform extraction, remove the residual chloroform of water-soluble part, use the moving phase constant volume to 25mL, mixing, with 0.22 μ m membrane filtration, analyze with HPLC.Adopt the chromatographic column of AgilentXDB-C18 (4.6 * 250mm, 5 μ m), flow velocity is adjusted to 0.6mL/min, detects wavelength 220nm, 30 ℃ of column temperatures, sample size 5 μ L.Referring to Fig. 2.No. 3 peaks and No. 4 peaks are the yang gum characteristic peak among the figure.
Get each 10g of propolis that mixes 10%, 20% and 30% yang gum, use 80mL 95% ethanol ultrasonic dissolution respectively, centrifugal, filter, remove ethanol with boulton process, add low amounts of water, use chloroform extraction, remove the residual chloroform of water-soluble part, with the moving phase constant volume to 25mL, mixing with 0.22 μ m membrane filtration, is analyzed with HPLC.Adopt the chromatographic column of Agilent XDB-C18 (4.6 * 250mm, 5 μ m), flow velocity is adjusted to 0.6mL/min, detects wavelength 220nm, 30 ℃ of column temperatures, sample size 5 μ L.See Fig. 3, Fig. 4 and Fig. 5 respectively.The characteristic peak that No. 1 peak and No. 2 peaks are propolis among the figure, and the characteristic peak that No. 3 and No. 4 peaks are yang gum.
According to said method, measured propolis respectively, yang gum and the propolis finger-print that mixes 10%, 20% and 30% yang gum, wherein No. 1 peak and No. 2 peaks are the propolis characteristic peak in the collection of illustrative plates, be not contain in the yang gum, and No. 3 peaks and No. 4 peaks are the yang gum characteristic peak, do not have this two peaks in the propolis.Mix in the propolis of 10%, 20% and 30% yang gum and all can detect 1,2,3 and No. 4 characteristic peak simultaneously.Therefore, can differentiate in propolis and yang gum and the propolis whether be mixed with yang gum with this.
Embodiment 2:
Get propolis 10g, centrifugal with 80mL 95% ethanol ultrasonic dissolution, filter, obtain propolis solution, remove ethanol with boulton process, add low amounts of water, use chloroform extraction, remove the residual chloroform of water-soluble part, to 25mL, mixing with 0.22 μ m membrane filtration, is analyzed with HPLC with the moving phase constant volume.Adopt the chromatographic column of AgilentXDB-C18 (4.6 * 250mm, 5 μ m), flow velocity is adjusted to 0.5mL/min, detects wavelength 213nm, 25 ℃ of column temperatures, sample size 2 μ L.
Get yang gum 2.5g,, filter, obtain yang gum solution with 80mL 95% ethanol ultrasonic dissolution, remove ethanol with boulton process, add low amounts of water, use chloroform extraction, remove the residual chloroform of water-soluble part, to 25mL, mixing with 0.22 μ m membrane filtration, is analyzed with HPLC with the moving phase constant volume.Adopt the chromatographic column of AgilentXDB-C18 (4.6 * 250mm, 5 μ m), flow velocity is adjusted to 0.5mL/min, detects wavelength 213nm, 25 ℃ of column temperatures, sample size 2 μ L.
According to said method, measured the finger-print of propolis (Fig. 6) and yang gum (Fig. 7) respectively, wherein No. 1 peak and No. 2 peaks are the propolis characteristic peak among Fig. 6, are not contain in the yang gum; And No. 3 peaks and No. 4 peaks are the yang gum characteristic peak among Fig. 7, are not contain in the propolis.Therefore, can differentiate propolis and yang gum with this.
Embodiment 3:
Get propolis 10g,, filter, obtain propolis solution with 80mL 95% ethanol ultrasonic dissolution, remove ethanol with boulton process, add low amounts of water, use chloroform extraction, remove the residual chloroform of water-soluble part, to 25mL, mixing with 0.22 μ m membrane filtration, is analyzed with HPLC with the moving phase constant volume.Adopt the chromatographic column of Agilent XDB-C18 (4.6 * 250mm, 5 μ m), flow velocity is adjusted to 1mL/min, detects wavelength 268nm, 30 ℃ of column temperatures, sample size 10 μ L.
Get yang gum 2.5g,, filter, obtain yang gum solution with 80mL 95% ethanol ultrasonic dissolution, remove ethanol with boulton process, add low amounts of water, use chloroform extraction, remove the residual chloroform of water-soluble part, to 25mL, mixing with 0.22 μ m membrane filtration, is analyzed with HPLC with the moving phase constant volume.Adopt the chromatographic column of AgilentXDB-C18 (4.6 * 250mm, 5 μ m), flow velocity is adjusted to 1mL/min, detects wavelength 268nm, 30 ℃ of column temperatures, sample size 10 μ L.
According to said method, measured the finger-print of propolis (Fig. 8) and yang gum (Fig. 9) respectively, wherein No. 1 peak and No. 2 peaks are the propolis characteristic peak among Fig. 8, are not contain in the yang gum; And No. 3 peaks and No. 4 peaks are the yang gum characteristic peak among Fig. 9, are not contain in the propolis.Therefore, can differentiate propolis and yang gum with this.
Embodiment 4:
Get propolis 10g,, filter, obtain propolis solution with 80ml 95% ethanol ultrasonic dissolution, remove ethanol with boulton process, add low amounts of water, use chloroform extraction, remove the residual chloroform of water-soluble part, to 25ml, mixing is used the 0.22um membrane filtration, analyzes with HPLC with the moving phase constant volume.Adopt the chromatographic column of AgilentXDB-C18 (4.6 * 250mm, 5 μ m), flow velocity is adjusted to 0.6ml/min, detects wavelength 265nm, 28 ℃ of column temperatures, sample size 10 μ L.
Get yang gum 2.5g,, filter, obtain yang gum solution with 80ml 95% ethanol ultrasonic dissolution, remove ethanol with boulton process, add low amounts of water, use chloroform extraction, remove the residual chloroform of water-soluble part, to 25ml, mixing with 0.22 μ m membrane filtration, is analyzed with HPLC with the moving phase constant volume.Adopt the chromatographic column of AgilentXDB-C18 (4.6 * 250mm, 5 μ m), flow velocity is adjusted to 0.6ml/min, detects wavelength 265nm, 28 ℃ of column temperatures, sample size 10 μ L.
According to said method, measured the finger-print of propolis (Figure 10) and yang gum (Figure 11) respectively, wherein No. 1 peak and No. 2 peaks are the propolis characteristic peak among Figure 10, are not contain in the yang gum; And No. 3 peaks and No. 4 peak yang gum characteristic peaks among Figure 11 are not contain in the propolis.Therefore, can differentiate propolis and yang gum with this.
Embodiment 5:
Get propolis 10g,, filter, obtain propolis solution with 80ml 95% ethanol ultrasonic dissolution, remove ethanol with boulton process, add low amounts of water, use chloroform extraction, remove the residual chloroform of water-soluble part, to 25ml, mixing with 0.22 μ m membrane filtration, is analyzed with HPLC with the moving phase constant volume.Adopt the chromatographic column of Agilent XDB-C18 (4.6 * 250mm, 5 μ m), flow velocity is adjusted to 0.8ml/min, detects wavelength 220nm, 26 ℃ of column temperatures, sample size 8 μ L.
Get yang gum 2.5g,, filter, obtain yang gum solution with 80ml 95% ethanol ultrasonic dissolution, remove ethanol with boulton process, add low amounts of water, use chloroform extraction, remove the residual chloroform of water-soluble part, to 25ml, mixing with 0.22u m membrane filtration, is analyzed with HPLC with the moving phase constant volume.Adopt the chromatographic column of AgilentXDB-C18 (4.6 * 250mm, 5 μ m), flow velocity is adjusted to 0.8ml/min, detects wavelength 220nm, 26 ℃ of column temperatures, sample size 8 μ L.
According to said method, measured the finger-print of propolis (Figure 12) and yang gum (Figure 13) respectively, wherein No. 1 peak and No. 2 peaks are the propolis characteristic peak among Figure 12, are not contain in the yang gum; And No. 3 peaks and No. 4 peaks are the yang gum characteristic peak among Figure 13, are not contain in the propolis.Therefore, can differentiate propolis and yang gum with this.
Claims (4)
1. the discrimination method of propolis and yang gum, realize by following scheme:
(1) with propolis, yang gum and the propolis that is mixed with yang gum respectively with the conventional ultrasonic Extraction of 95% ethanol, centrifugal, filter, obtain propolis respectively and extract the propolis that solution, yang gum extracts solution and be mixed with yang gum and extract solution,
(2) remove ethanol in each extract respectively, add chloroform extraction, remove the chloroform phase, repeatedly several times, stay water, wave the chloroform of clean aqueous phase, use the moving phase constant volume with separating funnel,
(3) analyze with rp-hplc, obtain propolis, yang gum respectively and be mixed with the chromatogram of the propolis of yang gum,
(4) with propolis, yang gum carries out discrimination ratio with the chromatogram that is mixed with the propolis of yang gum, thereby judges the true and false of propolis;
The condition of the described liquid chromatograph of step (3) is: flow velocity is 0.5～1.5mL/min, and the detection wavelength is 213～220nm, and sample size is 2～10 μ L, selects 4.6 * 250mm for use, the Agilent EclipseXDB-C18 chromatographic column of 5 μ m, and column temperature is 25～30 ℃.
2. the discrimination method of a kind of propolis according to claim 1 and yang gum is characterized in that, the volumetric ratio of the described moving phase of step (2) is methyl alcohol: 0.5%H
3PO
4=14: 86.
3. the discrimination method of a kind of propolis according to claim 1 and yang gum is characterized in that, the described detection wavelength of step (3) is selected 260～270nm for use.
4. the application of the discrimination method of described a kind of propolis of claim 1 and yang gum in differentiating propolis and yang gum.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910101428.4A CN101620208B (en) | 2009-08-06 | 2009-08-06 | Identification method for propolis and poplar gum |
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| CN200910101428.4A CN101620208B (en) | 2009-08-06 | 2009-08-06 | Identification method for propolis and poplar gum |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101871919A (en) * | 2010-05-21 | 2010-10-27 | 浙江大学 | Method for distinguishing bee glue and poplar glue |
| CN106770763A (en) * | 2016-12-21 | 2017-05-31 | 浙江海洋大学 | A kind of discrimination method of propolis and yang gum |
| CN108459038A (en) * | 2017-12-13 | 2018-08-28 | 江苏中谱检测有限公司 | The nuclear-magnetism finger print method that the willow type propolis true and false quickly differentiates |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001021548A (en) * | 1999-07-06 | 2001-01-26 | Kawasaki Steel Corp | Analytical method and device for free fluorine in solution containing hydrofluoric acid |
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2009
- 2009-08-06 CN CN200910101428.4A patent/CN101620208B/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101871919A (en) * | 2010-05-21 | 2010-10-27 | 浙江大学 | Method for distinguishing bee glue and poplar glue |
| CN106770763A (en) * | 2016-12-21 | 2017-05-31 | 浙江海洋大学 | A kind of discrimination method of propolis and yang gum |
| CN106770763B (en) * | 2016-12-21 | 2020-04-21 | 浙江海洋大学 | Method for identifying propolis and poplar gum |
| CN108459038A (en) * | 2017-12-13 | 2018-08-28 | 江苏中谱检测有限公司 | The nuclear-magnetism finger print method that the willow type propolis true and false quickly differentiates |
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| CN101620208B (en) | 2014-04-16 |
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